Methods of transdifferentiation and methods of use thereof

ABSTRACT

Disclosed herein is a method for manufacturing a population of human insulin producing cells from non-pancreatic β-cells, wherein the resulting insulin producing cells have increased insulin content, or increased glucose regulated secretion of insulin, or a combination of both.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/098,050, filed on Dec. 30, 2014, which is incorporated in itsentirety herein by reference.

FIELD OF THE INVENTION

The disclosure presented herein provides a method for large-scaleproduction of human insulin producing cells, wherein the insulinproducing cells comprise transdifferentiated non-pancreatic β-cell cellsthat produce insulin in a glucose regulated manner.

BACKGROUND OF THE INVENTION

The beta-cells of the islets of Langerhans in the pancreas secreteinsulin in response to factors such as amino acids, glyceraldehyde, freefatty acids, and, most prominently, glucose. The capacity of normalislet beta-cells to sense a rise in blood glucose concentration and torespond to elevated levels of glucose by secreting insulin is criticalto the control of blood glucose levels. Increased insulin secretion inresponse to a glucose load prevents hyperglycemia in normal individualsby stimulating glucose uptake into peripheral tissues, particularlymuscle and adipose tissue.

Individuals in whom islet beta-cells function is impaired suffer fromdiabetes. Insulin-dependent diabetes mellitus, or IDDM (also known asJuvenile-onset or Type I diabetes), represents approximately 10% of allhuman diabetes. IDDM is distinct from non-insulin dependent diabetes(NIDDM) in that only IDDM involves specific destruction of the insulinproducing beta cells of the islets of Langerhans. The destruction ofbeta-cells in IDDM appears to be a result of specific autoimmune attack,in which the patient's own immune system recognizes and destroys thebeta-cells, but not the surrounding alpha-cells (glucagon producing) ordelta-cells (somatostatin producing) that comprise the islet.

Treatment options for IDDM are centered on self-injection of insulin,which is an inconvenient and imprecise solution. Thus the development ofnew therapeutic strategies is highly desirable. The possibility of isletor pancreas fragment transplantation has been investigated as a meansfor permanent insulin replacement. Current methodologies use eithercadaverous material or porcine islets as transplant substrates. However,significant problems to overcome are the low availability of donortissue, the variability and low yield of islets obtained viadissociation, and the enzymatic and physical damage that may occur as aresult of the isolation process. In addition, there are issues of immunerejection and current concerns with xenotransplantation using porcineislets.

It is clear that there remains a critical need to establish alternativesto the treatment of diabetes by self-injection of insulin. While stemcell research has shown promise in this regard, there has not been greatsuccess. There is a need for improved procedures for isolating,culturing, and transdifferentiating non-pancreatic cells to be used inthe treatment of diabetes. The methods disclosed herein compriselarge-scale production of transdifferentiated non-beta pancreatic cellsthat secrete insulin. These transdifferentiated cells may be used intransplant therapies, obviating the need for the numerousself-injections of insulin, now required for the treatment of diabetes.

SUMMARY OF THE INVENTION

In one aspect, disclosed herein is a method of manufacturing apopulation of human insulin producing cells, the method comprising thesteps of: obtaining adult human liver tissue; processing said livertissue to recover primary adult human primary liver cells; propagatingand expanding said primary adult human liver cells to a predeterminednumber of cells; transdifferentiating said expanded cells; andharvesting said transdifferentiated expanded culture; therebymanufacturing said population of human insulin producing cells having anincreased insulin content, or increased glucose regulated secretion, orany combination thereof, compared with control non-transdifferentiatedliver cells.

In a related aspect, greater than 70% of said population of humaninsulin producing cells expresses endogenous PDX-1. In a further relatedaspect the cells expressing PDX-1 also express endogenous NeuroD1 orMafA, or any combination thereof. In yet another related aspect, lessthan 5% of the population expressing PDX-1 expresses albumin and alpha-1anti-trypsin.

In a related aspect, the increased insulin content of the cells producedcomprises an at least 5% increase compared with said control cells thatare not transdifferentiated.

In another aspect, the liver tissue is obtained from a subject sufferingfrom pancreatic or from insulin dependent diabetes. In a related aspect,the population of human insulin producing cells is autologous for apatient in need of such an insulin therapy. In another related aspect,the population of human insulin producing cells is allogeneic for apatient in need of such an insulin therapy.

In a related aspect, the method comprises propagating and expanding saidliver cells through a series of sub-cultivation steps up to aproduction-bioreactor system. In another related aspect, the bioreactorsystem comprises a single bioreactor or multiple bioreactors. In anotherrelated aspect, the bioreactor comprises a single use bioreactor, amulti-use bioreactor, a closed system bioreactor, or an open systembioreactor, or any combination thereof. In a further related aspect, thetransdifferentiating of said expanded cells comprisestransdifferentiation through a series of bioreactor systems.

In a related aspect, the transdifferentiating comprises: infecting saidexpanded cells with an adenoviral vector comprising a nucleic acidencoding a human PDX-1 polypeptide, said infecting at a first timeperiod; infecting said expanded cells of (a) with an adenoviral vectorcomprising a nucleic acid encoding a human NeuroD1 polypeptide or Pax4polypeptide, said infecting at a second time period; and infecting saidexpanded cells of (b) with an adenoviral vector comprising a nucleicacid encoding a human MafA polypeptide, said infecting at a third timeperiod.

In another related aspect, the transdifferentiating comprises: infectingsaid expanded cells with an adenoviral vector comprising a nucleic acidencoding a human PDX-1 polypeptide and encoding a second pancreatictranscription factor polypeptide, said infecting at a first time period;and infecting said expanded cells of (a) with an adenoviral vectorcomprising a nucleic acid encoding a human MafA polypeptide, saidinfecting at a second time period. In a further related aspect, thesecond pancreatic transcription factor is selected from NeuroD1 andPax4.

In another related aspect, the method further comprises enriching saidprimary adult human liver cells for cells predisposed totransdifferentiation. In a further related aspect, the predisposed cellscomprise pericentral liver cells. In yet another related aspect, thepredisposed cells comprise cells comprising: an active Wnt-signalingpathway; a capability of activating the glutamine synthetase responseelement (GSRE); increased expression of HOMER1, LAMP3, ITGA6, DCBLD2,THBS1, VAMP4, or BMPR2, or any combination thereof; decreased expressionof ABCB1, ITGA4, ABCB4, or PRNP, or any combination thereof; or anycombination thereof.

In another related aspect, the method further comprises treating theprimary adult human liver cell population with lithium, wherein saidtreated population is enriched in cells predisposed totransdifferentiation. In another related aspect, treating with lithiumoccurs prior to transdifferentiation.

In one aspect, disclosed herein is a population of human insulinproducing cells manufactured by a method comprising the steps of:obtaining adult human liver tissue; processing said liver tissue torecover primary adult human primary liver cells; propagating andexpanding said primary adult human liver cells to a predetermined numberof cells; transdifferentiating said expanded cells; and harvesting saidtransdifferentiated expanded culture; wherein said population of humaninsulin producing cells have an increased insulin content or increasedglucose regulated insulin secretion, or any combination thereof,compared with control non-transdifferentiated liver cells.

In another related aspect, greater than 70% of the population of humaninsulin producing cells expresses endogenous PDX-1. In a further relatedaspect, the cells expressing PDX-1 also express endogenous NeuroD1 orMafA, or any combination thereof. In yet another related aspect, lessthan 5% of the population expressing PDX-1 expresses albumin and alpha-1anti-trypsin.

In another related aspect, the population of human insulin producingcells comprises an increased insulin content comprising an at least 5%increase compared with said control cells.

In another related aspect, the population of human insulin producingcells is for use in a cell-based therapy for a patient suffering frompancreatitis or from insulin dependent diabetes. In a further relatedaspect, the cells are autologous or allogeneic with the patient.

In another aspect, disclosed herein is a composition comprising apopulation of human insulin producing cells, and a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as transdifferentiated non-beta pancreaticcells having the phenotype and function of pancreatic cells and methodsof manufacturing the same is particularly pointed out and distinctlyclaimed in the concluding portion of the specification. Thetransdifferentiated non-beta pancreatic cells having the phenotype andfunction of pancreatic cells, however, both as to organization andmethod of operation, together with objects, features, and advantagesthereof, may best be understood by reference to the following detaileddescription when read with the accompanying drawings in which:

FIGS. 1A-1D show that PDX-1 expression in human liver cells in vitroinduces gradual activation of pancreatic hormone expression. (FIG. 1A)Insulin (INS); (FIG. 1B) glucagon (GCG); (FIG. 1C) somatostatin (SST);and (FIG. 1D) other pancreas-specific transcription factors (“pTFs”)(NKX6.1, ISL1, PAX4, MAFA, NeuroD1, NeuroG3). The results werenormalized to β-actin gene expression within the same cDNA sample andare presented as the mean±SE of the relative expression versus controlvirus treated cells on the same day. n≧4 in two independent experiments(*p<0.05, **p<0.01).

FIGS. 2A-2D show that ectopic co-expression of pancreatic transcriptionfactors (pTFs) PDX-1, Pax4, and MafA in human liver cells in vitropromotes (pro)insulin secretion, compared to that induced by each of thepTFs alone. (FIG. 2A) Immunofluorescence (IF) staining shows expressionof pTFs: PDX-1 (left panel), Pax4 (middle left panel), MafA (middleright panel) and a merge of the 3 pTFs (right panel), with arrowsindicating cells expressing all three pTFs. (FIG. 2B) Luciferase assayinsulin promoter activation by the indicated pTFs; β-gal was used as acontrol. Results are expressed as Relative Light Unit (RLU)/mg protein.Each data point represents the mean±SE of at least two independentexperiments, *p<0.05, **p<0.01 in comparison to control virus treatedcells, (n>4). (FIG. 2C) Immunofluorescence staining showsinsulin-positive cells after ectopic expression of the indicated pTFs.Original magnification×20. Quantification of IF staining in table(right). The percent of insulin-positive cells was calculated bycounting at least 500 positive cells from at least two independentexperiments. (FIG. 2D) Insulin secretion after incubation with theindicated concentrations of glucose was detected by radioimmunoassay.*p<0.05, n>12 in five independent experiments. The significancerepresents the differences between triple infection and all othertreatments.

FIGS. 3A-3E show the effects of concerted and sequential expression ofpTFs PDX-1, Pax4, and MafA on pancreatic β-cell maturation. (FIG. 3A) Aschematic demonstrating the order of infection of pTFs (treatments B-E)or control virus (Ad-CMV-β-gal, treatment A). (FIG. 3B)Immunofluorescence staining for insulin: treatment B (left panel),treatment C (middle panel), treatment D (right panel). Originalmagnification is at ×20. Quantification of staining (percent) isindicated below each image. The percent of insulin positive cells werecalculated by counting at least 1000 positive cells from at least twoindependent experiments. (FIG. 3C) Insulin and (FIG. 3D) C-peptidesecretion after incubation with the indicated concentration of glucosewas measured by radioimmunoassay. Infection treatments are indicated onthe X-axis and explained in FIG. 3A. *p<0.05, **p<0.01, compared tocontrol virus treated cells; n>12 in 5 independent experiments. (FIG.3E) Expression levels of the indicated endogenous pancreas-specifictranscription factors after the indicated treatments (X-axis) weremeasured by RT-PCR. CT values are normalized to β-actin gene expressionwithin the same cDNA sample. Results are presented as relative levels ofthe mean+SE of the relative expression versus control virus treatedcells, *p<0.05 n>8 in 4 independent experiments. The arrow points thespecific decrease in Isl-1 expression level under treatment C.

FIGS. 4A-4C show three graphs demonstrating transdifferentiationefficiency, indicating hierarchical sequential order of infection(treatment C) is most efficient. (FIG. 4A) Insulin promoter activationwas measured by luciferase assay after the indicated infectiontreatments. Results are expressed as Relative Light Unit (RLU)/mgprotein. Each data point represents the mean±SE of at least twoindependent experiments, *P<0.05, **P<0.01, compared to control virustreated cells, (n>4). (FIG. 4B) Analysis of glucose transporter 2(GLUT2) expression levels by RT-PCR was performed after the indicatedinfection treatments. CT values are normalized to β-actin geneexpression within the same cDNA sample. Results are presented asrelative levels of the mean+SE compared to control virus treated cells.*P<0.05, compared to control virus treated cells n>8 in 4 independentexperiments. (FIG. 4C) Expression levels of prohormone convertase 2(PC2; PCSK2) were determined by RT-PCR after the indicated infectiontreatments. CT values are normalized to β-actin gene expression withinthe same cDNA sample. Results are presented as relative levels of themean+SE compared to control virus treated cells **P<0.01, n>8 in 4independent experiments.

FIGS. 5A-5B show two graphs demonstrating C-peptide secretion afterhierarchical sequential order of infection (treatment C). (FIG. 5A)C-peptide secretion was measured by radioimmunoassay static incubationfor 15 min at 0, 5, 10, 15, 20 mM glucose in cells treated by the direct“hierarchical” sequential order (treatment C) *P<0.05, n>7 in 3independent experiments. (FIG. 5B) C-peptide secretion was measured byradioimmunoassay over 13 or 28 days in serum free media supplementedwith insulin, transferrin and selenium (ITS), before being analyzed forC-peptide secretion. *P<0.05,**P<0.01, n>5 in 2 independent experiments.The significance represents the differences compared to the standardprotocol (treatment C on day 6).

FIGS. 6A-6D present four graphs showing the individual role of the pTFsin the transdifferentiation process, using treatment C infection orderand exclusion of each pTF (C-PDX-1, exclusion of PDX-1; C-Pax4,exclusion of Pax4; and C-MafA, exclusion of MafA). (FIG. 6A) Insulinpromoter activation was measured by luciferase assay. Results arepresented mean±SE, *p<0.1, **p<0.05 compared to the direct“hierarchical” sequential infection order (treatment C), n>6 in threeindependent experiments. (FIG. 6B) C-peptide secretion after incubationfor 15 minutes with the indicated concentrations of glucose and measuredby radioimmunoassay. *=p<0.05, **=p<0.01 is compared to the direct“hierarchical” sequential infection order (C), n>6 in three independentexperiments. (FIG. 6C) Expression levels of pancreatic enzymes weremeasured by RT-PCR: glucose transporter 2 (GLUT2); glucokinase (GCK);and prohormone convertase (PCSK2). (FIG. 6D) Expression levels of theindicated endogenous pTFs were measured by RT-PCR. CT values arenormalized to β-actin gene expression within the same cDNA sample.Results are presented as relative levels of the mean+SE compared to“hierarchy sequential infection” treated liver cells. *p<0.05, **p<0.01,n>6 in three independent experiments.

FIGS. 7A-7C shows three graphs showing the effects of Isl1 expression onβ-cell maturation of transdifferentiated liver cells after infection by“hierarchical” sequential order (treatment C). (FIG. 7A) Expressionlevels of insulin were measured by RT-PCR. CT values are normalized toβ-actin gene expression within the same cDNA sample. Results arepresented as relative levels of the mean+SE compared to control virustreated cells. *P<0.05, n>6 in 3 independent experiments. (FIG. 7B)Insulin secretion was measured by radioimmunoassay. **P<0.01, n>6 andcompared to the direct “hierarchical” sequential infection order (C),n>6 in 3 independent experiments. (FIG. 7C) Expression level of glucosetransporter 2 (GLUT2) was measured by RT-PCR.

FIGS. 8A-8G shows the individual role of pTFs in promoting thedifferentiation of cells to produce glucagon (α-cells) and somatostatin(δ-cells) using hierarchical order of infection (treatment C) andexclusion of each pTF. Expression levels of pancreatic hormones glucagon(GCG) (FIGS. 8A and 8B) and somatostatin (SST) (FIGS. 8A and 8D) weredetermined by RT-PCR after the indicated infection treatments. (FIG. 8C)Expression levels of cell-specific transcription factors ARX and BRAIN4were also measured by RT-PCR for the indicated infection treatments.(FIG. 8E) Expression levels of somatostatin (SST) were determined byRT-PCR after additional infection with Isl1 (100 MOI). CT values (forFIGS. 8A, 8B, 8C, and 8D) are normalized to β-actin gene expressionwithin the same cDNA sample. Results are presented as relative levels ofthe mean+SE compared to control virus treated cells (FIG. 8A) or to“hierarchy sequential infection” treated liver cells (FIGS. 8B-8E).*P<0.05, **P<0.1, n>6 in 3 independent experiments. (FIG. 8F)Immunofluorescence staining for somatostatin after treatment C infection(left panel), and after treatment C infection with additional Isl1infection (right panel). Original magnification×20. (FIG. 8G)Immunofluorescence staining for somatostatin and insulin showing thatthe sequential administration of transcription factors in a directhierarchical manner results in increased maturation of thetransdifferentiated cells along the beta-like-pancreatic lineage

FIG. 9 shows a schematic representation of the proposed mechanism ofpancreatic transcription factor-induced transdifferentiation from liverto pancreas. The concerted expression of the three pTFs results inincreased number of transdifferentiated liver cells compared to each ofthe factor's individual effect (Treatment B). The sequentialadministration of transcription factors in a direct hierarchical mannerresults in increased maturation of the Transdifferentiated cells alongthe beta-like-pancreatic lineage (Treatment C).

FIGS. 10A-10D shows PDX-1-induced insulin producing cells' (IPCs)activation in mice in vivo is restricted to cells adjacent to thecentral veins that are characterized by glutamine synthetase (GS)expression Immunohistochemical analysis of Pdx-1 (FIG. 10A) and insulin(FIG. 10B) 14 days after Ad-CMV-PDX-1 administration. Arrows indicatepositive cells, mostly located at the proximity of central veins (cv).(FIGS. 10C and 10D) analysis of GS expression in human (FIG. 10C) andmice (FIG. 10D) livers indicating the expression of GS at the 1-2 celllayers adjacent to the central veins. Original magnification×400.

FIG. 11 shows glutamine synthetase response element (GSRE) contains Wntsignaling responding element-TCF-LEF binding site. A schematicpresentation of GSRE indicating the presence of TCF-LEF and STAT 5binding sites.

FIGS. 12A-12F shows that the GSRE targets subpopulation of human livercells in vitro. (FIGS. 12A and 12D) Schematic presentations ofAd-GSRE-TK-PDX-1 or GFP recombinant adenoviruses. Liver cells wereinfected with Ad-GSRE-TK-Pdx-1 (FIG. 12C) or with Ad-CMV-Pdx-1 (FIG.12B) Immunofluorescent analysis of PDX-1 expression indicated that 13±2%of the human liver cells infected by Ad-GSRE-TK-Pdx-1 (FIG. 12C) while70±12% of Ad-CMV-Pdx-1-treated cells (FIG. 12B) expressed the ectopicnuclear factor (rabbit anti-Pdx-1, generous gift from C. Wright, pink;FIGS. 12B and 12C, respectively) Similar results were obtained usingAd-GSRE-TK-eGFP; ˜15% of the cells were positive to eGFP (FIGS. 12E and12F). Ad-CMV-eGFP infection resulted in about 75-80% eGFP positive cellswithin 3-4 days (data not presented).

FIGS. 13A-13C show that the GSRE targets transdifferentiation-pronecells. Liver cells were infected with Ad-GSRE-TK-Pdx-1 (FIG. 13B) orwith Ad-CMV-Pdx-1 (FIG. 13A) for 5 days. (FIGS. 13A and 13B),immunofluorescent analysis of co-staining of insulin (Guinea piganti-insulin, Dako, green) and (Pdx-1 rabbit anti-Pdx-1, generous giftfrom C. Wright, pink). (FIG. 13C) Statistical analysis of activation ofinsulin in the treated cells; Ad-GSRE-TK-Pdx-1 activated insulinproduction in 50%, whereas Ad-CMV-Pdx-1 only in 5% of the Pdx-1-positivecells. Blue—DAPI, nuclear staining; original magnification×20.

FIGS. 14A-14E show in vitro lineage tracing for GSRE activating humancells. (FIG. 14A) A schematic presentation of the lentivirus vectors.(FIG. 14B) Adult human liver cells at passages 3-10 were infected withthe dual lentivirus system. Liver cells were imaged 10 days afterinfection for DsRed2 (red) or eGFP (green) fluorescence. (FIG. 14C) Thecells were sorted by a fluorescence-activated cell sorter (FACS; Ariacell sorter; Becton Dickinson, San Jose, Calif.) with a fluoresceinisothiocyanate filter (530/30 nm) for eGFP and a Pe-Texas Red filter(610/20 nm) for DsRed2. (FIGS. 14D and 14E). The separated cells werecultured separately for several passages (original magnification×10).

FIGS. 15A-15E show eGFP+ and DsRed2+ cells efficiently proliferate invitro with a similar rate of proliferation and similar infectioncapacity. The separate populations of cells were cultured separately for˜1 month. The proliferation rate of each group was analyzed (FIG. 15A)eGFP+(FIGS. 15B and 15C) and DsRed2+(FIGS. 15D and 15E) cells wereinfected with Ad-CMV-β-gal (FIGS. 15B and 15D) or with Ad-CMV-Pdx-1(FIGS. 15C and 15E) for 3 days. Immunofluorescent analysis usinganti-Pdx-1 (blue) indicated that almost 80% of both eGFP and DsRed2cells were infected by the adenovirus.

FIGS. 16A-16C shows eGFP+ cells respond more efficiently than DsRed2+cells to pTFs-induced transdifferentiation. The two groups weresimilarly treated with soluble factors and pTFs:Ad-Pdx-1+Ad-Pax-4+ad-MafA or a control virus (Ad-β-gal) for 6 days.β-cell-like characteristics and function were compared in the separatedgroups: (FIG. 16A) at the molecular level, insulin and glucagon geneexpression was studied by Quantitative real-time PCR compared to thecontrol-treated cells. Cultured pancreatic human islet cells (Passage 3)were used as a positive control. (FIGS. 16B and 16C) At the functionallevel, glucose-regulated insulin secretion was analyzed by staticincubations at low glucose concentrations followed by high glucoseconcentrations (2 mM and 17.5 mM glucose in Krebs-Ringer buffer (KRB),respectively). Insulin (FIG. 16B) and C-peptide (FIG. 16C) secretionwere measured using the human insulin radioimmunoassay kit (DPC; n≧8from 3 different experiments) or human C-peptide radioimmunoassay kit(Linco n≧8 from 3 different experiments. *P<0.01 compared to the DsRed2+cells, using Student's t-test analysis.

FIG. 17 shows higher transdifferentiation efficiency in eGFP+ populationis stable with increasing passages in culture. The two groupsproliferated separately after sorting and were similarly treated withpTFs (Ad-Pdx-1+Ad-Pax-4+Ad-MafA and soluble factors) after a fewpassages (5-7 passages post sorting) or a higher number of passages(10-12 passages post sorting). Regulated insulin secretion was analyzedby static incubations at low followed by high glucose concentrations (2mM and 17.5 mM glucose in KRB, respectively). Insulin secretion ismeasured using the human insulin radioimmunoassay kit (DPC; n≧6 from 2different experiments). No statistical significant differences weredetected between the low and high number of passages in both populationsof cells, suggesting a persistent tendency of eGFP tagged cells toundergo pTFs induced transdifferentiation along the β-cell lineage andfunction.

FIG. 18 shows differential gene expression profiles of eGFP+ and DsRed2+cells performed by microarray analyses and analyzed according to DAVIDBioinformatics Resources 6.7. Four Percent of the differential genesbelong to the Wnt signaling pathway.

FIG. 19 shows that active Wnt signaling promotes liver to pancreastransdifferentiation. Adult human liver cells were treated withAd-CMV-Pdx-1 and soluble factors, as previously reported, supplementedwith Wnt3A (50 ng/ml R&D or DKK3 (3 μg/ml R&D). After 5 days, insulinsecretion was analyzed by static incubations at low followed by highglucose concentrations (2 mM and 17.5 mM glucose in KRB, respectively).Insulin secretion is measured using the human insulin radioimmunoassaykit (DPC; n≧8 from 3 different experiments) and compared to untreatedcells (Cont). *p<0.01 compared to Ad-CMV-Pdx-1 alone, using Student'st-test analysis.

FIG. 20 shows that blocking the Wnt signaling pathway abolishes thetransdifferentiation of eGFP+ cells. eGFP cells were Ad-CMV-Pdx-1 or acontrol virus (Ad-CMV-β-gal) for 5 days supplemented with DKK3(Dickkopf-related protein 3) (0.5 μg/ml R&D). Pancreatic hormones geneexpression was studied by Quantitative real-time RT-PCR compared to thecontrol-treated cells.

FIGS. 21A-21C show eGFP+ cells express lower levels of APC and higherlevels of active β-catenin than DsRed2+ cells. (FIG. 21A) APC and DKK1expression is markedly increased in DsRed2+ cells. This may furthersuggest that these cells express higher levels of Wnt signaling pathwayrepressors compared with the eGFP+ cells. n≧6 from 2 differentexperiments *p<0.01 in DsRed2+ compared to eGFP+ cells, using Student'st-test analysis. (FIG. 21B) Western blot analysis using a specificantibody for activated β-catenin (anti-ABC clone 8E7, Millipore, 1:2000)in eGFP and DsRed2 positive cell extracts. β-actin (SC-1616, Santa Cruz,1:1000) was used as a normalizing protein. (FIG. 21C) Quantification ofthe β-catenin protein levels was performed using ImageJ 1.29× software.Activated β-actin (SC-1616, Santa Cruz, 1:1000) was used as anormalizing protein.

FIG. 22 present micrographs showing mesenchymal stem cells (MSC) aresusceptible to adenovirus infection. MSC were infected by increasing moiof Ad-GFP. Five days later, cells were visualized by fluorescentmicroscopy (magnification×4) Representative phase contrast morphology(left panel), and green fluorescence (left panel) of MSC infected byAd-CMV-GFP. Infection of MSC cells with 1000 MOI of Ad-GFP resulted inabout 20-60% positive cells (dependent on cell-lines), when liver cellsusually present 70-80% positive cells.

FIG. 23 shows a bar graph showing that MSC secreted insulin in aglucose-regulated manner Cells were examined for their ability toundergo transdifferentiation. Transdifferentiation was induced on MSC byinfecting cells with PDX1, NeuroD1 and MafA. On the sixth day of theexperiment, cells underwent secretion experiment and RIA for Insulindetection. Insulin secretion in a glucose-regulated manner was measuredby incubation for 15 min with 2 mM or 17.5 mM glucose in KRB.

FIGS. 24A-24B present the combined insulin secretion measurements ofnaïve and GS enriched populations of cells on day 6 of the experimentcomparing the effect of PAX4 versus NeuroD1. FIG. 24A presents a bargraph of insulin secretion in response to low (2 mM) and high (17 mM)concentrations of glucose as Nano grams insulin per million cells perhour (ng/10⁶/hr). FIG. 24B presents a bar graph of insulin secretion inresponse to low (2 mM) and high (17 mM) concentrations of glucose asNano grams insulin per hour (ng/hr).

FIGS. 25A-24D present the individual insulin secretion measurements ofnaïve and enriched populations of cells on day 6 of the experimentcomparing the effect of PAX4 versus NeuroD1. FIG. 25A (enriched for GSexpression) and FIG. 25C (Naïve) present bar graphs of insulin secretionin response to low (2 mM) and high (17 mM) concentrations of glucose asNano grams insulin per million cells per hour (ng/10⁶/hr). FIG. 25B(enriched for GS expression) and FIG. 25D (Naïve) presents a bar graphof insulin secretion in response to low (2 mM) and high (17 mM)concentrations of glucose as Nano grams insulin per hour (ng/hr).

FIGS. 26A-26C show insulin secretion measured on day 6 of the experimentfollowing incubation with 2 mM glucose (low concentration) or 17.5 mMglucose (high concentration). Results are presented as Nano gramsinsulin per million cells per hour (ng INS/10⁶/hr) for primary livercells obtained from human donors (FIG. 26A Muhammad, FIG. 26B Pedro, andFIG. 26C Leon).

FIG. 27 presents schematics of the human liver-derived cellamplification and transdifferentiation process indicating thepreclinical R&D process (Cell Culture Dish Process) and the clinicalprocess (Xpansion Bioreactor Process).

FIG. 28 presents a typical seed train and cell expansion profile ofhuman liver-derived primary cells from multi-tray Cell Stack (CS) 10plates to the XP-200 bioreactor. Dotted lines in green represent atarget in terms of numbers of cells required per patient (targetingdiabetes cell-based autologous therapy), wherein the target number shownis 1 billion cells per patient. PDL represents Population DoublingLimit. CS represents Cell Stack multitrays.

FIG. 29 presents Population Doubling Time (PDT) in days in the XP-50 andXP-200 bioreactors and in their control classic multi-tray supportcounterparts (CTL XP50 and CTL XP-200). The data is based on harvestedcell densities. The numbers in each bar represent the PDT.

FIG. 30 shows the regulation trend in the bioreactors (XP-50 and XP-200)for pH (green), DO (blue), and temperature (red). Dotted lines representthe set points and peaks were due to bioreactor disconnection fordifferent operations (for example, media exchange).

FIGS. 31A-31D present microscopic observations of cells within theXpansion bioreactor (FIGS. 31A and 31B) and control multi-tray systems(FIGS. 31C and 31D) before harvest on day 9. FIGS. 31A and 31C showcells from the Xpansion 50 bioreactor run, while FIG. 31B and FIG. 31Dshow cells from the Xpansion 200 bioreactor run.

FIG. 32 presents a liver cell-based autologous cell therapy schema,adapted from Cozar-Castellan and Stewart (2005) Proc Nat Acad Sci USA102(22): 7781-7782.

FIG. 33 presents a manufacturing process showing adult human primaryliver cells undergoing a 1,000-fold expansion beforetransdifferentiation and final quality assurance/quality control (QA/QC)testing.

FIG. 34 presents an overview of the autologous insulin-producing (AIP)cell manufacturing process. Steps include: Step 1—Obtaining liver tissue(e.g., a liver biopsy); Step 2—Processing of the tissue to recoverprimary liver cells; Step 3—Propagating the primary liver cells topredetermined cell number; Step 4—Transdifferentiation of the primaryliver cells; Step 5—Harvesting of the primary transdifferentiated livercells; and Step 6—testing the transdifferentiated cells for qualityassurance and quality control (i.e., safety, purity and potency).Optional steps include cryopreserving early passage primary liver cells,where in one embodiment an early passage is passage 1; thawingcryopreserved cells for use at a later date and storage oftransdifferentiated cells for use at a later date.

FIG. 35 presents the variability of cell density at harvest from cellsmanufactured during three individual runs, wherein the startingdensities are comparable.

FIGS. 36A and 36B presents bar graphs displaying typical results ofendogenous gene expression from populations of transdifferentiated humanprimary liver cells, the results showing an increase in endogenous ofpancreatic cell markers (PDX-1, NeuroD1, MafA, glucagon, andsomatostatin) compared with control untreated (non-transdifferentiated)cells.

FIG. 37 presents the results of testing for AIP cell product Potency(glucose regulated insulin secretion, assayed by ELISA).

FIG. 38 present a flowchart showing three different “2+1”transdifferentiation protocols, including protocols using multi-systembioreactors, for the production of human insulin producing cells fromnon-pancreatic cells, as shown here starting from liver cells. Theflowchart indicates target cell densities at seeding and plating postinfection, as well as the first infection comprising infecting withadeno viral vectors comprising DNA encoding PDX-1 and NeuroD1polypeptides, and the second infection comprising infecting with anadenoviral vector comprising DNA encoding MafA. In all, seeding toharvest occurs in about 8 days.

FIGS. 39A-39D present micrographs of cell densities at day 6 at the timeof second infection, including an image of untreated control cells.

FIGS. 40A-40B present micrographs of cell densities at day 6 at the timeof second infection from plates 3 (FIG. 40A) and 5 (FIG. 40B) of theXpansion-10 multi-system bioreactor.

FIGS. 41A-41D present micrographs of cell densities at day 8 at the timeof the final harvest, including an image of untreated control cells.

FIGS. 42A-42B present micrographs of cell densities at day 8 at the timeof final harvest from plates 3 (FIG. 42A) and 5 (FIG. 42B) of theXpansion-10 multi-system bioreactor.

FIG. 43 presents a bar graph showing the results of an insulin contentassays for cells produced using the “2+1” protocol (See FIG. 38),showing that transdifferentiation in a bioreactor system is not onlyfeasible, but yields human insulin producing cells wherein the cellshave increased insulin content compared with control untreated cells.

FIGS. 44A and 44B present the results of flow cytometry analysis ofexpanded and transdifferentiated liver cells. FIG. 44A shows arepresentative FACS plot of several mesenchymal stem cells (MSC)markers, gated on live cells. Markers shown include CD90, CD73, CD105,and CD44. The Negative cocktail includes hematopoietic markers. FIG. 44Bshows the frequency of the MSC markers at different cell passages, P12(12^(th) passage), P13 (13^(th) passage), P14 (14^(th) passage), and ininfected cells (P16_AdV infection).

FIGS. 45A-45C transduction efficiency of BP001 liver cells. FIG. 45Afluorescent micrographs, FIG. 45B FACS, and FIG. 45C Summary of FACSdata.

FIGS. 46A-46C show transduction efficiency of TS001 liver cells. FIG.46A fluorescent micrographs, FIG. 46B FACS, and FIG. 46C Summary of FACSdata.

FIG. 47 presents a bar graph of the relative expression levels ofcell-surface molecules in eGFP+ and DsRed2+ cells, listed in Table 2B ofExample 16.

FIGS. 48A-48C shows pre-existing WNT/β-catenin signal disposes cells toefficient transdifferentiation. WNT signaling was induced by Li for 48hours prior to transdifferentiation, which was then removed (Li day −2)or maintained (Li day −2 onward) throughout the transdifferentiationprotocol. Insulin secretion was measured by ELISA, in response to 17.5mM glucose stimulation. FIG. 48A bar graph shows fold increase ofinsulin following transdifferentiation without pre-treatment of lithium(left) and with pre-treatment of lithium 48 hours prior totransdifferentiation (right). (FIGS. 48B and 48C). Expression levels ofpancreatic genes Nkx6.1, Isl-1, and human PDX1 were measured byReal-Time PCR, and normalized to actin. Results are representative oftwo donors.

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements may be exaggeratedrelative to other elements for clarity.

DETAILED DESCRIPTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the non-pancreatictransdifferentiated human insulin producing cells having pancreatic cellphenotype and functions, and methods of manufacturing the same. In otherinstances, well-known methods, procedures, and components have not beendescribed in detail so as not to obscure the non-pancreatictransdifferentiated human insulin producing cells having pancreatic cellphenotype and functions, and methods of producing the same.

Transcription factors (TFs) have been shown to inducetransdifferentiation in numerous cell lineages. A skilled artisan wouldappreciate that the term “transdifferentiation” may encompass theprocess by which a first cell type loses identifying characteristics andchanges its phenotype to that of a second cell type without goingthrough a stage in which the cells have embryonic characteristics. Insome embodiments, the first and second cells are from different tissuesor cell lineages. In one embodiment, transdifferentiation involvesconverting a mature or differentiated cell to a different mature ordifferentiated cell. Specifically, lineage-specific transcriptionfactors (TFs) have been suggested to display instructive roles inconverting adult cells to endocrine pancreatic cells, neurons,hematopoietic cells and cardiomyocyte lineages, suggesting thattransdifferentiation processes occur in a wide spectrum of milieus. Inall transdifferentiation protocols, the ectopic TFs serve as ashort-term trigger to a potential wide, functional and irreversibledevelopmental process. Numerous studies suggested that ectopicexpression of individual TFs activate a desired alternate repertoire andfunction, in a process involved with the activation of additionalrelevant otherwise silent TFs. However, the time course, the relativelevels and the hierarchy, or order, of the induced TFs, remains unknown.

By exploiting the relative insufficiency of the endogenous transcriptionfactor (TFs) induction by introducing individual ectopic TFs, disclosedherein are methods of transdifferentiation as a sequential andtemporally controlled process that is affected by a hierarchical networkof TFs.

The human insulin producing cell product, and methods thereof of makingand producing this product, as disclosed herein are based on the findingthat TF-induced liver to pancreas transdifferentiation is a gradual andconsecutive process. Importantly, only sequential administration ofpancreatic TFs but not their concerted expression selectively driveslineage specification programs within the endocrine pancreas. Sequentialexpression of pancreatic TFs in a direct hierarchical mode has beenshown to be obligatory for transdifferentiated cell maturation along theβ-cell lineage. Specifically, a role for the pancreatic β-cell specifictranscription factor MafA has been identified in the final stage of thetransdifferentiation process. At this stage, MafA promotes thematuration of transdifferentiated liver cells along the β-cell lineage,in a process associated with Isl1 and somatostatin repression.Surprisingly, it was found that a 2+1 hierarchical method (PDX-1 andPax4 or NeuroD1, followed by MafA) was successful for selectivelydriving lineage specification towards a pancreatic phenotype andfunction within non-pancreatic cells.

The findings described herein suggest fundamental temporalcharacteristics of transcription factor-mediated transdifferentiationwhich could contribute to increasing the therapeutic merit of usingTF-induced adult cell reprogramming for treating degenerative diseasesincluding diabetes.

Pancreatic transcription factor (pTFs), such as Pdx-1, NeuroD1, Ngn-3and Pax4, activate liver to pancreas transdifferentiation andindividually induce amelioration of hyperglycemia in diabetic mice.Moreover, using an in vitro experimental system of adult human livercells, it was demonstrated that Pdx-1 activates the expression ofnumerous β-cell specific markers and induces glucose-regulated secretionof processed insulin. The induced process was associated with theexpression of numerous key endogenous pTFs and amelioration ofhyperglycemia was demonstrated upon transplantation of thetransdifferentiated adult human liver cells in diabetic mice. However,numerous other studies have indicated that using combinations of severalkey TFs markedly increases the reprogramming efficiency compared to thatinduced by the ectopic expression of individual TFs. This suggests apotential restricted capacity of the individual ectopic factors toactivate the endogenous complementing TFs to sufficient levels neededfor an efficient transdifferentiation process. Targeted disruption ortemporal mis-expression of pancreatic transcription factors duringpancreas organogenesis hampers pancreas development as well as isletcells differentiation and function. By exploiting the relativeinsufficiency of the endogenous TFs induction by individual ectopic TFs,the disclosure presented herein is related to transdifferentiation as asequential and temporally controlled process that is affected by ahierarchical network of TFs.

Pancreatic specification is initiated by the homeobox transcriptionfactor Pdx1, which is also required for β-cell function in adults. Theendocrine differentiation is then mediated by the basic helix-loop-helixfactor Ngn3. The paired homeobox factors Pax4 and Arx, have beenimplicated as key factors in the segregation of the different endocrinecell types. The final maturation along the β-cell lineage and functionis attributed to selective expression of MafA in β-cells in the adultpancreas.

Disclosed herein are methods and human insulin producing cells producedusing these methods, based in part on the surprising finding that humanliver cells can be directly transdifferentiated to produce an entirelydifferent cell type, pancreatic hormones producing cells including betacells. Application of select transcription factors in a temporallyregulated sequence induced the transdifferentiation of adult liver cellsto functional mature beta cells. The methods described herein solve theproblem of producing large populations of insulin-producing cells, orpancreatic beta cells, by providing methods for expanding andtransdifferentiating adult cells. The compositions comprising the selecttranscription factors or the generated population of transdifferentiatedpancreatic cells can be used for treating a pancreatic disorder usingthe methods described herein.

Previous efforts to transdifferentiate non-pancreatic cells topancreatic cells, such as beta cells, utilize either only onetranscription factor or the concerted or simultaneous administration ofmore than one pancreatic transcription factor. The methods disclosedherein provide for an ordered, sequential administration of specifictranscription factors at defined time points. Alternative methodsdisclosed herein, provide for a “two pTFs+one pTF” (2+1) combined andordered, sequential administration of specific transcription factors atdefined time points. Furthermore, the methods described hereinsubstantially increase the transdifferentiation efficiency compared tothat induced by each of the individual transcription factors alone.

Disclosed herein is a population of cells that possess increasedtransdifferentiation capacity. These cells are characterized by (1)potential cell membrane markers, (2) possessing the capacity to activateglutamine synthetase regulatory element (GSRE), and (3) by beinguniquely equipped with active Wnt-signaling. At least 30% of the cellsin the population are capable of activating GSRE. For example the cellsare endothelial cells, epithelial cells, mesenchymal cells, fibroblasts,or liver cells. In one embodiment, the cells are human cells. In someembodiments, the cells can be transdifferentiated along the pancreaticlineage to mature pancreatic cells with pancreatic function. In otherembodiments, the cells can be transdifferentiated along the neurallineage to neural cells.

Thus, methods disclosed herein solve the problem of previoustransdifferentiation or reprogramming protocols that often haverestricted efficiency. For example, although ectopic expression of keypancreatic transcription factors results in expression in each hostcell, only up to 15% of the cells are successfully transdifferentiatedto exhibit pancreatic function.

Further, disclosed herein are methods for isolating the population ofcells with enriched or increased transdifferentiation capacity. Forexample, one method for isolating these cells is by sorting out cellsthat activate GFP expression operatively linked to the glutaminesynthetase regulatory element, or a fragment thereof, thereby isolatingthose cells that can activate GSRE. The cells may be sorted by FACS andcan be propagated in culture, separately from the rest of the cells, forrapid expansion of the cells with enriched transdifferentiationcapacity. The population of cells with enriched capacity fortransdifferentiation is only a small proportion of the cells that makeup the tissue in vivo. For example, in a given tissue or population ofcells, the population of cells with enriched capacity fortransdifferentiation is only about less than 1%, 2%, 3%, 4%, 5%, about10%, about 15%, of the entire population of cells in a given tissue.Therefore, methods are disclosed herein for the isolation of said cellswith increased transdifferentiation capacity from cells that do not haveincreased transdifferentiation capacity. Accordingly, the enrichednon-pancreatic β-cells, disclosed herein have the advantage of a cellpopulation with a greater proportion of cells that have increasedtransdifferentiation capacity to increase the efficiency oftransdifferentiation to provide transdifferentiated cells for treatmentof various diseases or disorders.

It will be obvious to those skilled in the art that various changes andmodifications may be made to the methods described herein within thespirit and scope of the non-pancreatic β-cells transdifferentiationhuman insulin producing cell product, and methods of making a using saidproduct.

Methods of Producing Pancreatic Beta-Cells

Disclosed herein are methods for producing cells that exhibit a maturepancreatic beta cell phenotype by contacting mammalian non-pancreaticcells with pancreatic transcription factors, such as PDX-1, Pax-4,NeuroD1, and MafA, at specific time points. In some embodiments, themethods comprise contacting a mammalian non-pancreatic cell with PDX-1at a first time period; contacting the cells from the first step withPax-4 at a second time period; and contacting the cells from the secondstep with MafA at a third time period. In one embodiment, the methodscomprise contacting a mammalian non-pancreatic cell with PDX-1 at afirst time period; contacting the cells from the first step with NeuroD1at a second time period; and contacting the cells from the second stepwith MafA at a third time period. In another embodiment, the methodscomprise contacting a mammalian non-pancreatic cell with PDX-1 and asecond transcription factor at a first time period and contacting thecells from the first step with MafA at a second time period. In yet afurther embodiment, a second transcription factor is selected fromNeuroD1 and Pax4. In another embodiment, the transcription factorsprovided together with PDX-1 comprise Pax-4, NeuroD1, Ngn3, or Sox-9. Inanother embodiment, the transcription factors provided together withPDX-1 comprises Pax-4. In another embodiment, the transcription factorsprovided together with PDX-1 comprises NeuroD1. In another embodiment,the transcription factors provided together with PDX-1 comprises Ngn3.In another embodiment, the transcription factors provided together withPDX-1 comprises Sox-9.

In other embodiments, the methods comprise contacting a mammaliannon-pancreatic cell with PDX-1 at a first time period; contacting thecells from the first step with Ngn3 at a second time period; andcontacting the cells from the second step with MafA at a third timeperiod. In other embodiments, the methods comprise contacting amammalian non-pancreatic cell with PDX-1 at a first time period;contacting the cells from the first step with Sox9 at a second timeperiod; and contacting the cells from the second step with MafA at athird time period. In another embodiment, the methods comprisecontacting a mammalian non-pancreatic cell with PDX-1 and a secondtranscription factor at a first time period and contacting the cellsfrom the first step with MafA at a second time period, wherein a secondtranscription factor is selected from NeuroD1, Ngn3, Sox9, and Pax4.

In another embodiment, the methods comprise contacting a mammaliannon-pancreatic cell with PDX-1 and NeuroD1 at a first time period, andcontacting the cells from the first step with MafA at a second timeperiod. In another embodiment, the methods comprise contacting amammalian non-pancreatic cell with PDX-1 and Pax4 at a first timeperiod, and contacting the cells from the first step with MafA at asecond time period. In another embodiment, the methods comprisecontacting a mammalian non-pancreatic cell with PDX-1 and Ngn3 at afirst time period, and contacting the cells from the first step withMafA at a second time period. In another embodiment, the methodscomprise contacting a mammalian non-pancreatic cell with PDX-1 and Sox9at a first time period, and contacting the cells from the first stepwith MafA at a second time period.

In another embodiment, the cells are contacted with all three factors(PDX-1; NeuroD1 or Pax4 or Ngn3; and MafA) at the same time but theirexpression levels are controlled in such a way as to have them expressedwithin the cell at a first time period for PDX-1, a second time periodfor NeuroD1 or Pax4 or Ngn3; and a third time period for MafA. Thecontrol of expression can be achieved by using appropriate promoters oneach gene such that the genes are expressed sequentially, by modifyinglevels of mRNA, or by other means known in the art.

In one embodiment, the methods described herein further comprisecontacting the cells at, before, or after any of the above steps withthe transcription factor Sox-9.

In one embodiment, the first and second time periods are identicalresulting in contacting a cell population with two pTFs at a first timeperiod, wherein at least one pTF comprises pDX-1, followed by contactingthe resultant cell population with a third pTF at a second time period,wherein said third pTF is MafA.

In one embodiment, the second time period is at least 24 hours after thefirst time period. In an alternative embodiment, the second time periodis less than 24 hours after the first time period. In anotherembodiment, the second time period is about 1 hour after the first timeperiod, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, or about 12 hours after the first time period. Insome embodiments, the second time period can be at least 24 hours, atleast 48 hours, at least 72 hours, and at least 1 week or more after thefirst time period.

In another embodiment, the third time period is at least 24 hours afterthe second time period. In an alternative embodiment, the third timeperiod is less than 24 hours after the second time period. In anotherembodiment, the third time period is at the same time as the second timeperiod. In another embodiment, the third time period is about 1 hourafter the second time period, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about9 hours, about 10 hours, about 11 hours, or about 12 hours after thesecond time period. In other embodiments, the third time period can beat least 24 hours, at least 48 hours, at least 72 hours, and at least 1week or more after the second time period.

In one embodiment, the first, second, and third time periods areconcurrent resulting in contacting a cell population with three pTFs ata single time period, wherein at least one pTF comprises pDX-1, at leastone pTF comprises NeuroD1 or Pax4, and at least one pTF comprises MafA.In another embodiment, the first, second, and third time periods areconcurrent resulting in contacting a cell population with three pTFs ata single time period, wherein one pTF consists of pDX-1, one pTFconsists of NeuroD1 or Pax4, and one pTF consists of MafA.

In one embodiment, transcription factors comprise polypeptides, orribonucleic acids or nucleic acids encoding the transcription factorpolypeptides. In another embodiment, the transcription factor comprisesa polypeptide. In another embodiment, the transcription factor comprisesa nucleic acid sequence encoding the transcription factor. In anotherembodiment, the transcription factor comprises a Deoxyribonucleic acidsequence (DNA) encoding the transcription factor. In still anotherembodiment, the DNA comprises a cDNA. In another embodiment, thetranscription factor comprises a ribonucleic acid sequence (RNA)encoding the transcription factor. In yet another embodiment, the RNAcomprises an mRNA.

Transcription factors for use in the disclosure presented herein can bea polypeptide, ribonucleic acid or a nucleic acid. A skilled artisanwould appreciate that the term “nucleic acid” may encompass DNAmolecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA,microRNA or other RNA derivatives), analogs of the DNA or RNA generatedusing nucleotide analogs, and derivatives, fragments and homologsthereof. The nucleic acid molecule can be single-stranded ordouble-stranded. In one embodiment, the nucleic acid is a DNA. In otherembodiments the nucleic acid is mRNA. mRNA is particularly advantageousin the methods disclosed herein, as transient expression of PDX-1 issufficient to produce pancreatic beta cells. The polypeptide,ribonucleic acid or nucleic acid may be delivered to the cell by meansknown in the art including, but not limited to, infection with viralvectors, electroporation and lipofection.

In certain embodiments, transcription factors for use in the methodsdescribed herein are selected from the group consisting of PDX-1, Pax-4,NeuroD1, and MafA. In other embodiments, transcription factors for usein the methods described herein are selected from the group consistingof PDX-1, Pax-4, NeuroD1, MafA, Ngn3, and Sox9.

The homeodomain protein PDX-1 (pancreatic and duodenal homeobox gene-1),also known as IDX-1, IPF-1, STF-1, or IUF-1, plays a central role inregulating pancreatic islet development and function. PDX-1 is eitherdirectly or indirectly involved in islet-cell-specific expression ofvarious genes such as, for example insulin, glucagon, somatostatin,proinsulin convertase 1/3 (PC1/3), GLUT-2 and glucokinase. Additionally,PDX-1 mediates insulin gene transcription in response to glucose.Suitable sources of nucleic acids encoding PDX-1 include for example thehuman PDX-1 nucleic acid (and the encoded protein sequences) availableas GenBank Accession Nos. U35632 and AAA88820, respectively. In oneembodiment, the amino acid sequence of a PDX-1 polypeptide is set forthin SEQ ID NO: 4:

(SEQ ID NO: 4) MNGEEQYYAATQLYKDPCAFQRGPAPEFSASPPACLYMGRQPPPPPPHPFPGALGALEQGSPPDISPYEVPPLADDPAVAHLHHHLPAQLALPHPPAGPFPEGAEPGVLEEPNRVQLPFPWMKSTKAHAWKGQWAGGAYAAEPEENKRTRTAYTRAQLLELEKEFLFNKYISRPRRVELAVMLNLTERHIKIWFQNRRMKWKKEEDKKRGGGTAVGGGGVAEPEQDCAVTSGEELLALPPPPPPGGAVPPAAPVAAREGRLPPGLSASPQPSSVAPRRPQEPR.

In one embodiment, the nucleic acid sequence of a PDX-1 polynucleotideis set forth in SEQ ID NO: 5:

(SEQ ID NO: 5) ATGAACGGCGAGGAGCAGTACTACGCGGCCACGCAGCTTTACAAGGACCCATGCGCGTTCCAGCGAGGCCCGGCGCCGGAGTTCAGCGCCAGCCCCCCTGCGTGCCTGTACATGGGCCGCCAGCCCCCGCCGCCGCCGCCGCACCCGTTCCCTGGCGCCCTGGGCGCGCTGGAGCAGGGCAGCCCCCCGGACATCTCCCCGTACGAGGTGCCCCCCCTCGCCGACGACCCCGCGGTGGCGCACCTTCACCACCACCTCCCGGCTCAGCTCGCGCTCCCCCACCCGCCCGCCGGGCCCTTCCCGGAGGGAGCCGAGCCGGGCGTCCTGGAGGAGCCCAACCGCGTCCAGCTGCCTTTCCCATGGATGAAGTCTACCAAAGCTCACGCGTGGAAAGGCCAGTGGGCAGGCGGCGCCTACGCTGCGGAGCCGGAGGAGAACAAGCGGACGCGCACGGCCTACACGCGCGCACAGCTGCTAGAGCTGGAGAAGGAGTTCCTATTCAACAAGTACATCTCACGGCCGCGCCGGGTGGAGCTGGCTGTCATGTTGAACTTGACCGAGAGACACATCAAGATCTGGTTCCAAAACCGCCGCATGAAGTGGAAAAAGGAGGAGGACAAGAAGCGCGGCGGCGGGACAGCTGTCGGGGGTGGCGGGGTCGCGGAGCCTGAGCAGGACTGCGCCGTGACCTCCGGCGAGGAGCTTCTGGCGCTGCCGCCGCCGCCGCCCCCCGGAGGTGCTGTGCCGCCCGCTGCCCCCGTTGCCGCCCGAGAGGGCCGCCTGCCGCCTGGCCTTAGCGCGTCGCCACAGCCCTCCAGCGTCGCGCCTCGGCGGCCGCAGGAACCACGATGA.

Other sources of sequences for PDX-1 include rat PDX nucleic acid andprotein sequences as shown in GenBank Accession No. U35632 and AAA18355,respectively, and are incorporated herein by reference in theirentirety. An additional source includes zebrafish PDX-1 nucleic acid andprotein sequences are shown in GenBank Accession No. AF036325 andAAC41260, respectively, and are incorporated herein by reference intheir entirety.

Pax-4, also known as paired box 4, paired box protein 4, paired box gene4, MODY9 and KPD, is a pancreatic-specific transcription factor thatbinds to elements within the glucagon, insulin and somatostatinpromoters, and is thought to play an important role in thedifferentiation and development of pancreatic islet beta cells. In somecellular contexts, Pax-4 exhibits repressor activity. Suitable sourcesof nucleic acids encoding Pax-4 include for example the human Pax-4nucleic acid (and the encoded protein sequences) available as GenBankAccession Nos. NM_006193.2 and AAD02289.1, respectively.

MafA, also known as V-maf musculoaponeurotic fibrosarcoma oncogenehomolog A or RIPE3B1, is a beta-cell-specific and glucose-regulatedtranscriptional activator for insulin gene expression. MafA may beinvolved in the function and development of beta cells as well as in thepathogenesis of diabetes. Suitable sources of nucleic acids encodingMafA include for example the human MafA nucleic acid (and the encodedprotein sequences) available as GenBank Accession Nos. NM_201589.3 andNP_963883.2, respectively. In one embodiment, the amino acid sequence ofa MafA polypeptide is set forth in SEQ ID NO: 8:

(SEQ ID NO: 8) MAAELAMGAELPSSPLAIEYVNDFDLMKFEVKKEPPEAERFCHRLPPGSLSSTPLSTPCSSVPSSPSFCAPSPGTGGGGGAGGGGGSSQAGGAPGPPSGGPGAVGGTSGKPALEDLYWMSGYQHHLNPEALNLTPEDAVEALIGSGHHGAHHGAHHPAAAAAYEAFRGPGFAGGGGADDMGAGHHHGAHHAAHHHHAAHHHHHHHHHHGGAGHGGGAGHHVRLEERFSDDQLVSMSVRELNRQLRGFSKEEVIRLKQKRRTLKNRGYAQSCRFKRVQQRHILESEKCQLQSQVEQLKLEVGRLAKERDLYKEKYEKLAGRGGPGSAGGAGFPREPS PPQAGPGGAKGTADFFL.

In another embodiment, the nucleic acid sequence of a MafApolynucleotide is set forth in SEQ ID NO: 9:

(SEQ ID NO: 9) ATGGCCGCGGAGCTGGCGATGGGCGCCGAGCTGCCCAGCAGCCCGCTGGCCATCGAGTACGTCAACGACTTCGACCTGATGAAGTTCGAGGTGAAGAAGGAGCCTCCCGAGGCCGAGCGCTTCTGCCACCGCCTGCCGCCAGGCTCGCTGTCCTCGACGCCGCTCAGCACGCCCTGCTCCTCCGTGCCCTCCTCGCCCAGCTTCTGCGCGCCCAGCCCGGGCACCGGCGGCGGCGGCGGCGCGGGGGGCGGCGGCGGCTCGTCTCAGGCCGGGGGCGCCCCCGGGCCGCCGAGCGGGGGCCCCGGCGCCGTCGGGGGCACCTCGGGGAAGCCGGCGCTGGAGGATCTGTACTGGATGAGCGGCTACCAGCATCACCTCAACCCCGAGGCGCTCAACCTGACGCCCGAGGACGCGGTGGAGGCGCTCATCGGCAGCGGCCACCACGGCGCGCACCACGGCGCGCACCACCCGGCGGCCGCCGCAGCCTACGAGGCTTTCCGCGGCCCGGGCTTCGCGGGCGGCGGCGGAGCGGACGACATGGGCGCCGGCCACCACCACGGCGCGCACCACGCCGCCCACCACCACCACGCCGCCCACCACCACCACCACCACCACCACCATGGCGGCGCGGGACACGGCGGTGGCGCGGGCCACCACGTGCGCCTGGAGGAGCGCTTCTCCGACGACCAGCTGGTGTCCATGTCGGTGCGCGAGCTGAACCGGCAGCTCCGCGGCTTCAGCAAGGAGGAGGTCATCCGGCTCAAGCAGAAGCGGCGCACGCTCAAGAACCGCGGCTACGCGCAGTCCTGCCGCTTCAAGCGGGTGCAGCAGCGGCACATTCTGGAGAGCGAGAAGTGCCAACTCCAGAGCCAGGTGGAGCAGCTGAAGCTGGAGGTGGGGCGCCTGGCCAAAGAGCGGGACCTGTACAAGGAGAAATACGAGAAGCTGGCGGGCCGGGGCGGCCCCGGGAGCGCGGGCGGGGCCGGTTTCCCGCGGGAGCCTTCGCCGCCGCAGGCCGGTCCCGGCGGGGCCAAGGGCACGGCCGACTTCTTCCTG TAG

Neurog3, also known as neurogenin 3 or Ngn3, is a basic helix-loop-helix(bHLH) transcription factor required for endocrine development in thepancreas and intestine. Suitable sources of nucleic acids encodingNeurog3 include for example the human Neurog3 nucleic acid (and theencoded protein sequences) available as GenBank Accession Nos.NM_020999.3 and NP_066279.2, respectively.

NeuroD1, also known as Neuro Differentiation 1 or NeuroD, and beta-2(β2) is a Neuro D-type transcription factor. It is a basichelix-loop-helix transcription factor that forms heterodimers with otherbHLH proteins and activates transcription of genes that contain aspecific DNA sequence known as the E-box. It regulates expression of theinsulin gene, and mutations in this gene result in type II diabetesmellitus. Suitable sources of nucleic acids encoding NeuroD1 include forexample the human NeuroD1 nucleic acid (and the encoded proteinsequences) available as GenBank Accession Nos. NM_002500.4 andNP_002491.2, respectively.

In one embodiment, the amino acid sequence of a NeuroD1 polypeptide isset forth in SEQ ID NO: 6:

(SEQ ID NO: 6) MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLETMNAEEDSLRNGGEEEDEDEDLEEEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQPTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMSF DSHSHHERVMSAQLNAIFHD.

In another embodiment, the nucleic acid sequence of a NeuroD1polynucleotide is set forth in SEQ ID NO: 7.

(SEQ ID NO: 7) ATGACCAAATCGTACAGCGAGAGTGGGCTGATGGGCGAGCCTCAGCCCCAAGGTCCTCCAAGCTGGACAGACGAGTGTCTCAGTTCTCAGGACGAGGAGCACGAGGCAGACAAGAAGGAGGACGACCTCGAAGCCATGAACGCAGAGGAGGACTCACTGAGGAACGGGGGAGAGGAGGAGGACGAAGATGAGGACCTGGAAGAGGAGGAAGAAGAGGAAGAGGAGGATGACGATCAAAAGCCCAAGAGACGCGGCCCCAAAAAGAAGAAGATGACTAAGGCTCGCCTGGAGCGTTTTAAATTGAGACGCATGAAGGCTAACGCCCGGGAGCGGAACCGCATGCACGGACTGAACGCGGCGCTAGACAACCTGCGCAAGGTGGTGCCTTGCTATTCTAAGACGCAGAAGCTGTCCAAAATCGAGACTCTGCGCTTGGCCAAGAACTACATCTGGGCTCTGTCGGAGATCTCGCGCTCAGGCAAAAGCCCAGACCTGGTCTCCTTCGTTCAGACGCTTTGCAAGGGCTTATCCCAACCCACCACCAACCTGGTTGCGGGCTGCCTGCAACTCAATCCTCGGACTTTTCTGCCTGAGCAGAACCAGGACATGCCCCCGCACCTGCCGACGGCCAGCGCTTCCTTCCCTGTACACCCCTACTCCTACCAGTCGCCTGGGCTGCCCAGTCCGCCTTACGGTACCATGGACAGCTCCCATGTCTTCCACGTTAAGCCTCCGCCGCACGCCTACAGCGCAGCGCTGGAGCCCTTCTTTGAAAGCCCTCTGACTGATTGCACCAGCCCTTCCTTTGATGGACCCCTCAGCCCGCCGCTCAGCATCAATGGCAACTTCTCTTTCAAACACGAACCGTCCGCCGAGTTTGAGAAAAATTATGCCTTTACCATGCACTATCCTGCAGCGACACTGGCAGGGGCCCAAAGCCACGGATCAATCTTCTCAGGCACCGCTGCCCCTCGCTGCGAGATCCCCATAGACAATATTATGTCCTTCGATAGCCATTCACATCATGAGCGAGTCATGAGTGCCCAGCTCAATGCC ATATTTCATGATTAG.

Sox9 is a transcription factor that is involved in embryonicdevelopment. Sox9 has been particularly investigated for its importancein bone and skeletal development. SOX-9 recognizes the sequence CCTTGAGalong with other members of the HMG-box class DNA-binding proteins. Inthe context of the disclosure presented herein, the use of Sox9 may beinvolved in maintaining the pancreatic progenitor cell mass, eitherbefore or after induction of transdifferentiation. Suitable sources ofnucleic acids encoding Sox9 include for example the human Sox9 nucleicacid (and the encoded protein sequences) available as GenBank AccessionNos. NM_000346.3 and NP_000337.1, respectively.

Homology is, in one embodiment, determined by computer algorithm forsequence alignment, by methods well described in the art. For example,computer algorithm analysis of nucleic acid sequence homology mayinclude the utilization of any number of software packages available,such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST EnhancedAlignment Utility), GENPEPT and TREMBL packages.

In another embodiment, “homology” refers to identity to a sequenceselected from SEQ ID No: 4-9 of greater than 60%. In another embodiment,“homology” refers to identity to a sequence selected from SEQ ID No:1-76 of greater than 70%. In another embodiment, the identity is greaterthan 75%, greater than 78%, greater than 80%, greater than 82%, greaterthan 83%, greater than 85%, greater than 87%, greater than 88%, greaterthan 90%, greater than 92%, greater than 93%, greater than 95%, greaterthan 96%, greater than 97%, greater than 98%, or greater than 99%. Inanother embodiment, the identity is 100%. Each possibility represents aseparate embodiment of the disclosure presented herein.

In another embodiment, homology is determined via determination ofcandidate sequence hybridization, methods of which are well described inthe art (See, for example, “Nucleic Acid Hybridization” Hames, B. D.,and Higgins S. J., Eds. (1985); Sambrook et al., 2001, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y). For example methodsof hybridization may be carried out under moderate to stringentconditions, to the complement of a DNA encoding a native caspasepeptide. Hybridization conditions being, for example, overnightincubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA.

Protein and/or peptide homology for any amino acid sequence listedherein is determined, in one embodiment, by methods well described inthe art, including immunoblot analysis, or via computer algorithmanalysis of amino acid sequences, utilizing any of a number of softwarepackages available, via established methods. Some of these packages mayinclude the FASTA, BLAST, MPsrch or Scanps packages, and may employ theuse of the Smith and Waterman algorithms, and/or global/local or BLOCKSalignments for analysis, for example. Each method of determininghomology represents a separate embodiment of the disclosure presentedherein.

The cell can be any cell that is capable of producing pancreatichormones, e.g., bone marrow muscle, spleen, kidney, blood, skin,pancreas, or liver. In one embodiment, the cell is a non-pancreaticcell. In another embodiment, the cell is a non-pancreatic β-cell. In oneembodiment, the cells are capable of functioning as a pancreatic islet,i.e., store, process and secrete pancreatic hormones. In anotherembodiment, secretion is glucose regulated.

In another embodiment, glucose regulated insulin secretion comprises atleast 0.001 pg insulin/10⁶ cells/hour in response to high glucoseconcentrations. In another embodiment, glucose regulated insulinsecretion comprises at least 0.002 pg insulin/10⁶ cells/hour in responseto high glucose concentrations. In another embodiment, glucose regulatedinsulin secretion comprises at least 0.003 pg insulin/10⁶ cells/hour inresponse to high glucose concentrations. In another embodiment, glucoseregulated insulin secretion comprises at least 0.005 pg insulin/10⁶cells/hour in response to high glucose concentrations. In anotherembodiment, glucose regulated insulin secretion comprises at least 0.007pg insulin/10⁶ cells/hour in response to high glucose concentrations. Inanother embodiment, glucose regulated insulin secretion comprises atleast 0.01 pg insulin/10⁶ cells/hour in response to high glucoseconcentrations. In another embodiment, glucose regulated insulinsecretion comprises at least 0.5 pg insulin/10⁶ cells/hour in responseto high glucose concentrations. In another embodiment, glucose regulatedinsulin secretion comprises at least 1 pg insulin/10⁶ cells/hour inresponse to high glucose concentrations. In another embodiment, glucoseregulated insulin secretion comprises at least 5 pg insulin/10⁶cells/hour in response to high glucose concentrations. In anotherembodiment, glucose regulated insulin secretion comprises at least 10 pginsulin/10⁶ cells/hour in response to high glucose concentrations. Inanother embodiment, glucose regulated insulin secretion comprises atleast 50 pg insulin/10⁶ cells/hour in response to high glucoseconcentrations. In another embodiment, glucose regulated insulinsecretion comprises at least 100 pg insulin/10⁶ cells/hour in responseto high glucose concentrations. In another embodiment, glucose regulatedinsulin secretion comprises at least 500 pg insulin/10⁶ cells/hour inresponse to high glucose concentrations. In another embodiment, glucoseregulated insulin secretion comprises at least 1 ng insulin/10⁶cells/hour in response to high glucose concentrations. In anotherembodiment, glucose regulated insulin secretion comprises at least 5 nginsulin/10⁶ cells/hour in response to high glucose concentrations. Inanother embodiment, glucose regulated insulin secretion comprises atleast 10 ng insulin/10⁶ cells/hour in response to high glucoseconcentrations. In another embodiment, glucose regulated insulinsecretion comprises at least 50 ng insulin/10⁶ cells/hour in response tohigh glucose concentrations. In another embodiment, glucose regulatedinsulin secretion comprises at least 100 ng insulin/10⁶ cells/hour inresponse to high glucose concentrations. In another embodiment, glucoseregulated insulin secretion comprises at least 500 ng insulin/10⁶cells/hour in response to high glucose concentrations. In anotherembodiment, glucose regulated insulin secretion comprises at least 1 μginsulin/10⁶ cells/hour in response to high glucose concentrations. Inanother embodiment, glucose regulated insulin secretion comprises atleast 5 μg insulin/10⁶ cells/hour in response to high glucoseconcentrations. In another embodiment, glucose regulated insulinsecretion comprises at least 10 μg insulin/10⁶ cells/hour in response tohigh glucose concentrations. In another embodiment, glucose regulatedinsulin secretion comprises at least 50 μg insulin/10⁶ cells/hour inresponse to high glucose concentrations. In another embodiment, glucoseregulated insulin secretion comprises at least 100 μg insulin/10⁶cells/hour in response to high glucose concentrations.

In another embodiment, the pancreatic hormone comprises insulin, whichmay be secreted upon an extracellular trigger. In another embodiment,the cell is a liver cell. In additional embodiments, the cell istotipotent or pluripotent. In alternative embodiments the cell is ahematopoietic stem cell, embryonic stem cell or preferably a hepaticstem cell. In other embodiments, the cell is an induced pluripotent stemcells.

In one embodiment, the source of a cell population disclosed here in isa human source. In another embodiment, the source of a cell populationdisclosed here in is an autologous human source relative to a subject inneed of insulin therapy. In another embodiment, the source of a cellpopulation disclosed here in is an allogeneic human source relative to asubject in need of insulin therapy.

In certain embodiments, the cell is a mesenchymal stem cell, also knownas a mesenchymal stromal cell, (MSC) such as a MSC derived from, livertissue, adipose tissue, bone marrow, skin, placenta, umbilical cord,Wharton's jelly or cord blood. By “umbilical cord blood” or “cord blood”is meant to refer to blood obtained from a neonate or fetus, mostpreferably a neonate and preferably refers to blood which is obtainedfrom the umbilical cord or the placenta of newborns. These cells can beobtained according to any conventional method known in the art. MSC aredefined by expression of certain cell surface markers including, but notlimited to, CD105, CD73 and CD90 and ability to differentiate intomultiple lineages including osteoblasts, adipocytes and chondroblasts.MSC can be obtained from tissues by conventional isolation techniquessuch as plastic adherence, separation using monoclonal antibodies suchas STRO-1 or through epithelial cells undergoing anepithelial-mesenchymal transition (EMT).

A skilled artisan would appreciate that the term “adipose tissue-derivedmesenchymal stem cells” may encompass undifferentiated adult stem cellsisolated from adipose tissue and may also be term “adipose stem cells”,having all the same qualities and meanings. These cells can be obtainedaccording to any conventional method known in the art.

A skilled artisan would appreciate that the term, “placental-derivedmesenchymal stem cells” may encompass undifferentiated adult stem cellsisolated from placenta and may be referred to herein as “placental stemcells”, having all the same meanings and qualities.

The cell population that is exposed to, i.e., contacted with, thecompounds (i.e. PDX-1, Pax-4, MafA, NeuroD1 and/or Sox-9 polypeptides ornucleic acid encoding PDX-1, Pax-4, MafA, NeuroD1 and/or Sox-9polypeptides) can be any number of cells, i.e., one or more cells, andcan be provided in vitro, in vivo, or ex vivo. The cell population thatis contacted with the transcription factors can be expanded in vitroprior to being contacted with the transcription factors. The cellpopulation produced exhibits a mature pancreatic beta cell phenotype.These cells can be expanded in vitro by methods known in the art priorto transdifferentiation and maturation along the β-cell lineage, andprior to administration or delivery to a patient or subject in needthereof.

The subject is, in one embodiment, a mammal. The mammal can be, e.g., ahuman, non-human primate, mouse, rat, dog, cat, horse, or cow.

In some embodiments, the transcription factor is a polypeptide, such asPDX-1, Pax-4, MafA, NeuroD1 or Sox-9, or combination thereof and isdelivered to a cell by methods known in the art. For example, thetranscription factor polypeptide is provided directly to the cells ordelivered via a microparticle or nanoparticle, e.g., a liposomalcarrier.

In some embodiments, the transcription factor is a nucleic acid. Forexample, the nucleic acid encodes a PDX-1, Pax-4, MafA, NeuroD1 or Sox-9polypeptide. The nucleic acid encoding the transcription factor, or acombination of such nucleic acids, can be delivered to a cell by anymeans known in the art. In some embodiments, the nucleic acid isincorporated in an expression vector or a viral vector. In oneembodiment, the viral vector is an adenovirus vector. In anotherembodiment, an adenoviral vector is a first generation adenoviral (FGAD)vector. In another embodiment, an FGAD is unable in integrate into thegenome of a cell. In another embodiment, a FGAD comprises an E1-deletedrecombinant adenoviral vector. In another embodiment, a FGAD providetransient expression of encoded polypeptides.

The expression or viral vector can be introduced to the cell by any ofthe following: transfection, electroporation, infection, ortransduction. In other embodiments the nucleic acid is mRNA and it isdelivered for example by electroporation. In one embodiment, methods ofelectroporation comprise flow electroporation technology. For example,in another embodiment, methods of electroporation comprise use of aMaxCyte electroporation system (MaxCyte Inc. Georgia USA).

In certain embodiments, the manufactured population of human insulinproducing cells comprises a reduction of liver phenotypic markers. Inone embodiment, there is a reduction of expression of albumin, alpha-1anti-trypsin, or a combination thereof. In another embodiment, less than5% of the cell population expressing endogenous PDX-1 expresses albuminand alpha-1 anti-trypsin. In another embodiment, less than 10%, 9%, 8%,7%, 6%, 4%, 3%, 2%, or 1% of the cell population expressing endogenousPDX-1 expresses albumin and alpha-1 anti-trypsin.

Cell Populations Predisposed for Transdifferentiation

The disclosure presented herein provides liver derived cell populationsthat are predisposed for transdifferentiation. The cell populations maybe useful in the methods of producing pancreatic beta cells describedherein. Cells that are predisposed for transdifferentiation of thedisclosure presented herein may also be referred to as having increasedor enriched transdifferentiation capacity. By “increasedtransdifferentiation capacity” is meant that when the cell population ofthe disclosure presented herein is subjected to a differentiationprotocol (i.e. introduction of a pancreatic transcription factor),greater than 15%, greater than 20%, greater than 30%, greater than 40%,greater than 50%, greater than 60%, greater than 70% or greater than 80%of the cells may differentiate to an alternate cell type. In oneembodiment, a population of endothelial cells, epithelial cells,mesenchymal cells, fibroblasts, or liver cells with increasedtransdifferentiation capacity may be differentiated to mature pancreaticcells or mature neural cells (transdifferentiation).

In another embodiment, cell populations that are predisposed fortransdifferentiation have the capability of activating the glutaminesynthetase response element (GSRE). For example, in the cell populationsof the disclosure presented herein, at least 2%, at least 3%, at least4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80% orat least 90% of the cells in the population are capable of activatingGSRE. In one embodiment, at least 30% of the cells in the population arecapable of activating GSRE. Glutamine synthetase is an enzymepredominantly expressed in the brain, kidneys and liver, and plays anessential role in the metabolism of nitrogen by catalyzing thecondensation of glutamate and ammonia to form glutamine. Glutaminesynthetase is, for example, uniquely expressed in pericentral livercells and astrocytes in the brain. Data presented herein indicate thatcells that demonstrate activation of GSRE provide a unique selectiveparameter for the isolation of cells predisposed fortransdifferentiation. In another embodiment, a predisposed population ofcells comprises pericentral liver cells.

Activation of GSRE can be measured by methods known to one of ordinaryskill in the art. For example, a recombinant adenovirus can be generatedcontaining the glutamine synthetase response element operatively linkedto a promoter and a reporter gene, such as a fluorescent protein. Thisrecombinant adenovirus with the GSRE-reporter can be introduced into aheterogeneous mixture of cells containing some proportion of cells thatare predisposed for transdifferentiation. Those cells that are competentfor activation of the GSRE will express the reporter gene, which can bedetected by methods known in the art, thereby identifying cellspredisposed for transdifferentiation.

A heterogeneous population of cells, in which those cells predisposedfor transdifferentiation are unknown, can be contacted with anadenoviral vector that contains the GSRE operatively linked to a minimalTK promoter and eGFP. The cells that activate the GSRE will express GFPand can be identified by various methods known in the art to detect GFPexpression. For example, separation of the GSRE-activated cells whichare predisposed for transdifferentiation from the cells that are notpredisposed for transdifferentiation can be achieved through FACsapparatus, sorter and techniques known to those ordinarily skilled inthe art (FIG. 14). The separated cells that are predisposed fortransdifferentiation can then be propagated or expanded in vitro.Results described herein demonstrate that passaging of the cellspredisposed for transdifferentiation for 5-12 passages or more retaintheir transdifferentiation capacity. For example, isolated liver cellspredisposed for transdifferentiation continue to produce and secreteinsulin in a glucose-dependent manner even after 12 passages in culture(FIG. 17).

In another embodiment, cell populations that are predisposed fortransdifferentiation also have active Wnt signaling pathways. Wntsignaling pathways play a significant role in stem cell pluripotency andcell fate during development, as well as body axis patterning, cellproliferation, and cell migration. Wnt signaling pathways are activatedby the binding of a Wnt-protein ligand to a Frizzled (Fz) familyreceptor (a G-coupled protein receptor), optionally activating aco-receptor protein, and the subsequent activation of a cytoplasmicprotein called Dishevelled (Dsh). In the canonical Wnt pathway,co-receptor LRP-5/6 is also activated and beta-catenin accumulates inthe cytoplasm and is eventually translocated into the nucleus to act asa transcriptional coactivator of TCF/LEF transcription factors. WithoutWnt signaling, a destruction complex that includes proteins such asadenomatosis polyposis coli (APC), Axin, protein phosphatase 2A (PP2A),glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α) targetsβ-catenin for ubiquitination and its subsequent degradation by theproteasome. However, activation of the Frizzled receptor by Wnt bindingcauses disruption of the destruction complex, thereby allowing β-cateninto accumulate.

Wnt signaling can also occur through noncanonical pathways that utilizedifferent co-receptor proteins and activate different downstreameffectors to, for example, regulate of the cytoskeleton, stimulate ofcalcium release from the endoplasmic reticulum, activate mTOR pathways,and regulate myogenesis.

One of ordinary skill in the art could readily use methods known in theart to determine the activation of Wnt signaling pathways. For example,cells that express Wnt3a, decreased levels of DKK1 or DKK3, decreasedlevels of APC, increased activated beta-catenin levels, or STATS bindingelements have active Wnt signaling pathways. DKK1, DKK3, and APC areknown inhibitors of Wnt signaling pathways. Other signaling effectorsthat indicate active Wnt signaling pathways are readily known in theart.

In one embodiment, methods disclosed further comprise treating theprimary adult human liver cell population with lithium, wherein saidtreated population is enriched in cells predisposed totransdifferentiation. In another embodiment, methods disclosed furthercomprise treating the primary adult human liver cell population withlithium, wherein said cells predisposed to transdifferentiation withinthe population have an increased predisposition following treatment withlithium. Thus, an enriched population of cells predisposed totransdifferentiation may be established by treating a primary adultpopulation of cells with lithium.

In one embodiment, a primary adult population of cells is treated with10 mM of lithium. In another embodiment, a primary adult population ofcells is treated with 1 mM of lithium. In one embodiment, a primaryadult population of cells is treated with between 1-10 mM of lithium. Inone embodiment, a primary adult population of cells is treated with 2 mMof lithium. In one embodiment, a primary adult population of cells istreated with 3 mM of lithium. In one embodiment, a primary adultpopulation of cells is treated with 4 mM of lithium. In one embodiment,a primary adult population of cells is treated with 5 mM of lithium. Inone embodiment, a primary adult population of cells is treated with 6 mMof lithium. In one embodiment, a primary adult population of cells istreated with 7 mM of lithium. In one embodiment, a primary adultpopulation of cells is treated with 8 mM of lithium. In one embodiment,a primary adult population of cells is treated with 9 mM of lithium. Inone embodiment, a primary adult population of cells is treated withabout 10-20 mM of lithium. In one embodiment, a primary adult populationof cells is treated with 15 mM of lithium. In one embodiment, a primaryadult population of cells is treated with 20 mM of lithium. In oneembodiment, a primary adult population of cells is treated with 10-50 mMof lithium. In one embodiment, a primary adult population of cells istreated with 10-100 mM of lithium.

In another embodiment, cells were treated prior to the time oftransdifferentiation (the first time period). In another embodiment,cells were treated 12 hours prior to transdifferentiation (the firsttime period). In another embodiment, cells were treated 24 hours priorto transdifferentiation (the first time period). In another embodiment,cells were treated 36 hours prior to transdifferentiation (the firsttime period). In another embodiment, cells were treated 48 hours priorto transdifferentiation (the first time period). In another embodiment,cells were treated 60 hours prior to transdifferentiation (the firsttime period). In another embodiment, cells were treated 72 hours priorto transdifferentiation (the first time period). In yet anotherembodiment, cells were treated at the time of transdifferentiation (thefirst time period).

In one embodiment, the cell populations used in methods disclosed hereinare predisposed for transdifferentiation to the pancreatic lineage,wherein the transdifferentiated cells exhibit pancreatic phenotype andfunction. For example, the transdifferentiated cells exhibit maturepancreatic beta cell phenotype and function, which includes, but is notlimited to, expression, production, and/or secretion of pancreatichormones. Pancreatic hormones can include, but are not limited to,insulin, somatostatin, glucagon, or islet amyloid polypeptide (IAPP).Insulin can be hepatic insulin or serum insulin. In one embodiment, theinsulin is a fully process form of insulin capable of promoting glucoseutilization, and carbohydrate, fat and protein metabolism. For example,the cells predisposed for transdifferentiation may encompass about 15%of all the cells in a heterogeneous in vitro primary human liver cellculture. When the cells ectopically express pTFs, greater than 5%, 10%,15%, 20%, 25%, 30%, 40%, 50% of the cells in culture produce insulin orsecrete C-peptide.

In one embodiment, cell populations that are predisposed fortransdifferentiation are located in close proximity to the central veinsof the liver, or are pericentral liver cells. As shown herein, althoughover 40-50% of liver cells that ectopically express pancreatictranscription factors, such as PDX-1, only a subset of cells producedinsulin upon pTF expression. These insulin-producing cells (IPCs) wereprimarily located close to the ventral veins, as shown by FIG. 10B.These cells are also characterized by expression of glutamine synthetaseand active Wnt signaling.

In another embodiment, the cell populations used in methods disclosedherein is predisposed for transdifferentiation to the neural lineage,wherein the transdifferentiated cells express neural markers, exhibitneural phenotype, or exhibit neural function. The transdifferentiatedcells can be neurons or glial cells.

In another embodiment, cells with increased predisposition fortransdifferentiation may be identified through specific cell surfacemarkers. For example, cells with increased levels of HOMER1, LAMPS orBMPR2 indicate cells with increased transdifferentiation capacity whencompared to cells without predisposition for transdifferentiation. Cellswith decreased levels of ABCB1, ITGA4, ABCB4, or PRNP indicate cellswith increased transdifferentiation capacity when compared to cellswithout predisposition for transdifferentiation. Any combination of thecell surface markers described can be used to distinguish a cellpopulation predisposed to transdifferentiation from a cell populationthat is not predisposed to transdifferentiation. Antibodies to thesecell surface markers are commercially available Immunoassay orimmunoaffinity techniques known in the art may be utilized todistinguish cells with increased transdifferentiation capacity fromthose cells without transdifferentiation capacity.

Use of the cell populations of the disclosure presented herein toproduce cells that exhibit pancreatic cell phenotypes provide certainadvantages over differentiating heterogeneous populations ofnon-pancreatic cells to produce cells that exhibit pancreatic cellphenotypes. Previous studies that describe expressing a pancreatictranscription factor (pTF) such as PDX-1 in a heterogeneous populationof non-pancreatic cells (i.e., liver cells) show that at best, only 15%of the PDX-1-expressing cells are competent for producing insulin.Therefore, only 15% of the cells were successfully differentiated to amature pancreatic beta cell capable of producing and secretingpancreatic hormones. In contrast, introducing pTFs into the cellpopulations of the disclosure presented herein results in at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%of the cells are differentiated to a mature pancreatic beta cellphenotype, for example, produce insulin, glucagon, and/or secretec-peptide. In one embodiment, when the cells of the cell population ofthe disclosure presented herein express a pancreatic transcriptionfactor, at least 30% of the cells produce insulin or secrete C-peptide.

Methods of Transdifferentiation

The disclosure presented herein also provides methods for utilizing thecell populations with increased transdifferentiation capacity to producecells that exhibit a mature differentiated cell type, where thedifferentiated cell has a different phenotype from the starting cellpopulation. For example, a population of cells with increasedtransdifferentiation capacity (i.e. epithelial cells, fibroblasts orliver cells) can be differentiated to cells along the pancreatic orneural lineage to exhibit mature differentiated pancreatic or neuralcell phenotypes. Any means known in the art for differentiating cells topancreatic or neural lineage can be utilized.

In one embodiment, the cell population predisposed fortransdifferentiation may be differentiated along the neural lineagethrough the expression of neural transcription factors. Suitable neuraltranscription factors are known in the art. In other embodiments, thecell population of the disclosure presented herein may be differentiatedto mature neural cells through contacting the cells with variouscytokines, growth factors, or other agents known in the art todifferentiate cells to the neural lineage. The differentiated neuralcells may express neural markers, exhibit a neural phenotype (i.e.,neural gene expression profile), or exhibit neural function. Thedifferentiated cells can be neurons or glial cells.

In another embodiment, the cell population predisposed fortransdifferentiation may be differentiated along the pancreatic lineagethrough the expression of pancreatic transcription factors. Thepancreatic transcription factors are, for example, PDX-1, Pax-4, MafA,NeuroD1, or a combination thereof. Methods for producing pancreatic betacells are described in U.S. Pat. No. 6,774,120 and U.S. Publication No.2005/0090465, the contents of which are incorporated by reference intheir entireties.

In another embodiment, the cell population predisposed fortransdifferentiation may be differentiated along the pancreatic lineagethrough the methods described herein.

Pancreatic Beta-Cell Phenotypes

The methods provided herein produce cells with a mature pancreatic betacell phenotype or function. A skilled artisan would appreciate that theterm “pancreatic beta cell phenotype or function” may encompass cellsthat display one or more characteristics typical of pancreatic betacells, i.e. pancreatic hormone production, processing, storage insecretory granules, hormone secretion, activation of pancreatic genepromoters, or characteristic beta cell gene expression profile. Hormonesecretion includes nutritionally and/or hormonally regulated secretion.In one embodiment, the cells produced exhibit at least one pancreaticbeta cell phenotype or function, as described herein.

The pancreatic hormone can be for example, insulin, glucagon,somatostatin or islet amyloid polypeptide (IAPP). Insulin can be hepaticinsulin or serum insulin. In another embodiment the pancreatic hormoneis hepatic insulin. In an alternative embodiment the pancreatic hormoneis serum insulin (i.e., a fully processed form of insulin capable ofpromoting, e.g., glucose utilization, carbohydrate, fat and proteinmetabolism).

In some embodiments the pancreatic hormone is in the “prohormone” form.In other embodiments the pancreatic hormone is in the fully processedbiologically active form of the hormone. In other embodiments thepancreatic hormone is under regulatory control i.e., secretion of thehormone is under nutritional and hormonal control similar toendogenously produced pancreatic hormones. For example, in oneembodiment disclosed herein, the hormone is under the regulatory controlof glucose.

The pancreatic beta cell phenotype can be determined for example bymeasuring pancreatic hormone production, i.e., insulin, somatostatin orglucagon protein mRNA or protein expression. Hormone production can bedetermined by methods known in the art, i.e. immunoassay, Western blot,receptor binding assays or functionally by the ability to amelioratehyperglycemia upon implantation in a diabetic host. Insulin secretioncan also be measured by, for example, C-peptide processing andsecretion. In another embodiment, high-sensitivity assays may beutilized to measure insulin secretion. In another embodiment,high-sensitivity assays comprise an enzyme-linked immunosorbent assay(ELISA), a mesoscale discovery assay (MSD), or an Enzyme-LinkedImmunoSpot assay (ELISpot), or an assay known in the art.

In some embodiments, the cells may be directed to produce and secreteinsulin using the methods specified herein. The ability of a cell toproduce insulin can be assayed by a variety of methods known to those ofordinary skill in the art. For example, insulin mRNA can be detected byRT-PCR or insulin may be detected by antibodies raised against insulin.In addition, other indicators of pancreatic differentiation include theexpression of the genes Isl-1, Pdx-1, Pax-4, Pax-6, and Glut-2. Otherphenotypic markers for the identification of islet cells are disclosedin U.S. 2003/0138948, incorporated herein in its entirety.

The pancreatic beta cell phenotype can be determined for example bypromoter activation of pancreas-specific genes. Pancreas-specificpromoters of particular interest include the promoters for insulin andpancreatic transcription factors, i.e. endogenous PDX-1. Promoteractivation can be determined by methods known in the art, for example byluciferase assay, EMSA, or detection of downstream gene expression.

In some embodiments, the pancreatic beta-cell phenotype can also bedetermined by induction of a pancreatic gene expression profile. Askilled artisan would appreciate that the term “pancreatic geneexpression profile” may encompass a profile to include expression of oneor more genes that are normally transcriptionally silent innon-endocrine tissues, i.e., a pancreatic transcription factor,pancreatic enzymes or pancreatic hormones. Pancreatic enzymes are, forexample, PCSK2 (PC2 or prohormone convertase), PC1/3 (prohormoneconvertase 1/3), glucokinase, glucose transporter 2 (GLUT-2).Pancreatic-specific transcription factors include, for example, Nkx2.2,Nkx6.1, Pax-4, Pax-6, MafA, NeuroD1, NeuroG3, Ngn3, beta-2, ARX, BRAIN4and Isl-1.

Induction of the pancreatic gene expression profile can be detectedusing techniques well known to one of ordinary skill in the art. Forexample, pancreatic hormone RNA sequences can be detected in, e.g.,Northern blot hybridization analyses, amplification-based detectionmethods such as reverse-transcription based polymerase chain reaction orsystemic detection by microarray chip analysis. Alternatively,expression can be also measured at the protein level, i.e., by measuringthe levels of polypeptides encoded by the gene. In a specific embodimentPC1/3 gene or protein expression can be determined by its activity inprocessing prohormones to their active mature form. Such methods arewell known in the art and include, e.g., immunoassays based onantibodies to proteins encoded by the genes, or HPLC of the processedprohormones.

In some embodiments, the cells exhibiting a mature beta-cell phenotypegenerated by the methods described herein may repress at least one geneor the gene expression profile of the original cell. For example, aliver cell that is induced to exhibit a mature beta-cell phenotype mayrepress at least one liver-specific gene. One skilled in the art couldreadily determine the liver-specific gene expression of the originalcell and the produced cells using methods known in the art, i.e.measuring the levels of mRNA or polypeptides encoded by the genes. Uponcomparison, a decrease in the liver-specific gene expression wouldindicate that transdifferentiation has occurred.

In certain embodiments, the transdifferentiated cells disclosed hereincomprise a reduction of liver phenotypic markers. In one embodiment,there is a reduction of expression of albumin, alpha-1 anti-trypsin, ora combination thereof. In another embodiment, less than 5% of the cellpopulation expressing endogenous PDX-1 expresses albumin and alpha-1anti-trypsin. In another embodiment, less than 10%, 9%, 8%, 7%, 6%, 4%,3%, 2%, or 1% of the transdifferentiated cells expressing endogenousPDX-1 expresses albumin and alpha-1 anti-trypsin.

Methods of Treating a Pancreatic Disorder

The disclosure presented herein discloses methods for use in treating,i.e., preventing or delaying the onset or alleviating a symptom of apancreatic disorder in a subject. For example, the pancreatic disorderis a degenerative pancreatic disorder. The methods disclosed herein areparticularly useful for those pancreatic disorders that are caused by orresult in a loss of pancreatic cells, e.g., islet beta cells, or a lossin pancreatic cell function.

Common degenerative pancreatic disorders include, but are not limitedto: diabetes (e.g., type I, type II, or gestational) and pancreaticcancer. Other pancreatic disorders or pancreas-related disorders thatmay be treated by using the methods disclosed herein are, for example,hyperglycemia, pancreatitis, pancreatic pseudocysts or pancreatic traumacaused by injury. Additionally, individuals whom have had apancreatectomy are also suitable to treatment by the disclosed methods

Diabetes is a metabolic disorder found in three forms: type 1, type 2and gestational. Type 1, or IDDM, is an autoimmune disease; the immunesystem destroys the pancreas' insulin-producing beta cells, reducing oreliminating the pancreas' ability to produce insulin. Type 1 diabetespatients must take daily insulin supplements to sustain life. Symptomstypically develop quickly and include increased thirst and urination,chronic hunger, weight loss, blurred vision and fatigue. Type 2 diabetesis the most common, found in 90 percent to 95 percent of diabetessufferers. It is associated with older age, obesity, family history,previous gestational diabetes, physical inactivity and ethnicity.Gestational diabetes occurs only in pregnancy. Women who developgestational diabetes have a 20 percent to 50 percent chance ofdeveloping type 2 diabetes within five to 10 years.

A subject suffering from or at risk of developing diabetes is identifiedby methods known in the art such as determining blood glucose levels.For example, a blood glucose value above 140 mg/dL on at least twooccasions after an overnight fast means a person has diabetes. A personnot suffering from or at risk of developing diabetes is characterized ashaving fasting sugar levels between 70-110 mg/dL.

Symptoms of diabetes include fatigue, nausea, frequent urination,excessive thirst, weight loss, blurred vision, frequent infections andslow healing of wounds or sores, blood pressure consistently at or above140/90, HDL cholesterol less than 35 mg/dL or triglycerides greater than250 mg/dL, hyperglycemia, hypoglycemia, insulin deficiency orresistance. Diabetic or pre-diabetic patients to which the compounds areadministered are identified using diagnostic methods know in the art.

Hyperglycemia is a pancreas-related disorder in which an excessiveamount of glucose circulates in the blood plasma. This is generally aglucose level higher than (200 mg/dl). A subject with hyperglycemia mayor may not have diabetes.

Pancreatic cancer is the fourth most common cancer in the U.S., mainlyoccurs in people over the age of 60, and has the lowest five-yearsurvival rate of any cancer. Adenocarcinoma, the most common type ofpancreatic cancer, occurs in the lining of the pancreatic duct;cystadenocarcinoma and acinar cell carcinoma are rarer. However, benigntumors also grow within the pancreas; these include insulinoma—a tumorthat secretes insulin, gastrinoma—which secretes higher-than-normallevels of gastrin, and glucagonoma—a tumor that secretes glucagon.

Pancreatic cancer has no known causes, but several risks, includingdiabetes, cigarette smoking and chronic pancreatitis. Symptoms mayinclude upper abdominal pain, poor appetite, jaundice, weight loss,indigestion, nausea or vomiting, diarrhea, fatigue, itching or enlargedabdominal organs. Diagnosis is made using ultrasound, computedtomography scan, magnetic resonance imaging, ERCP, percutaneoustranshepatic cholangiography, pancreas biopsy or blood tests. Treatmentmay involve surgery, radiation therapy or chemotherapy, medication forpain or itching, oral enzymes preparations or insulin treatment.

Pancreatitis is the inflammation and autodigestion of the pancreas. Inautodigestion, the pancreas is destroyed by its own enzymes, which causeinflammation. Acute pancreatitis typically involves only a singleincidence, after which the pancreas will return to normal. Chronicpancreatitis, however, involves permanent damage to the pancreas andpancreatic function and can lead to fibrosis. Alternately, it mayresolve after several attacks. Pancreatitis is most frequently caused bygallstones blocking the pancreatic duct or by alcohol abuse, which cancause the small pancreatic ductules to be blocked. Other causes includeabdominal trauma or surgery, infections, kidney failure, lupus, cysticfibrosis, a tumor or a scorpion's venomous sting.

Symptoms frequently associated with pancreatitis include abdominal pain,possibly radiating to the back or chest, nausea or vomiting, rapidpulse, fever, upper abdominal swelling, ascites, lowered blood pressureor mild jaundice. Symptoms may be attributed to other maladies beforebeing identified as associated with pancreatitis.

Method of Treating a Neurological Disorders

The disclosure presented herein also provides methods for treating asubject with a neurological disease or disorder, such as aneurodegenerative disease disorder. The population of cells describedherein is useful for treating a subject with a neurological disease ordisorder that is characterized by loss of neural cells or neuralfunction, by way of replenishing the degenerated or nonfunctional cells.Neurodegenerative diseases that may be treated using the methodsdescribed herein include, but are not limited to, Parkinson's disease,Parkinsonian disorders, Alzheimer's disease, Huntington's disease,amyotrophic lateral sclerosis, Lewy body disease, age-relatedneurodegeneration, neurological cancers, and brain trauma resulting fromsurgery, accident, ischemia, or stroke. The population of cellsdescribed herein can be differentiated to a neural cell population withneural function, and the differentiated neural cell population may beadministered to a subject with a neurological disease or disorder.

Recombinant Expression Vectors and Host Cells

Another embodiment disclosed herein, pertains to vectors. In oneembodiment, a vector used in methods disclosed herein comprises anexpression vector. In another embodiment, an expression vector comprisesa nucleic acid encoding a PDX-1, Pax-4, NeuroD1 or MafA protein, orother pancreatic transcription factor, such as Ngn3, or derivatives,fragments, analogs, homologs or combinations thereof. In someembodiments, the expression vector comprises a single nucleic acidencoding any of the following transcription factors: PDX-1, Pax-4,NeuroD1, Ngn3, MafA, or Sox-9 or derivatives or fragments thereof. Insome embodiments, the expression vector comprises two nucleic acidsencoding any combination of the following transcription factors: PDX-1,Pax-4, NeuroD1, Ngn3, MafA, or Sox-9 or derivatives or fragmentsthereof. In a yet another embodiment, the expression vector comprisesnucleic acids encoding PDX-1 and NeuroD1. In a still another embodiment,the expression vector comprises nucleic acids encoding PDX-1 and Pax4.In another embodiment, the expression vector comprises nucleic acidsencoding MafA.

A skilled artisan would appreciate that the term “vector” encompasses anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichencompasses a linear or circular double stranded DNA loop into whichadditional DNA segments can be ligated. Another type of vector is aviral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. A skilled artisan wouldappreciate that the terms “plasmid” and “vector” may be usedinterchangeably having all the same qualities and meanings. In oneembodiment, the term “plasmid” is the most commonly used form of vector.However, the disclosure presented herein is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, lentivirus, adenoviruses andadeno-associated viruses), which serve equivalent functions.Additionally, some viral vectors are capable of targeting a particularcells type either specifically or non-specifically.

The recombinant expression vectors disclosed herein comprise a nucleicacid disclosed herein, in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, a skilled artisan would appreciate that the term “operablylinked” may encompass nucleotide sequences of interest linked to theregulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). A skilled artisan would appreciate that term “regulatorysequence” may encompass promoters, enhancers and other expressioncontrol elements (e.g., polyadenylation signals). Such regulatorysequences are described, for example, in Goeddel; GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Regulatory sequences include those that direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosethat direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors disclosed here may be introduced into host cellsto thereby produce proteins or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., PDX-1,Pax-4, MafA, NeuroD1 or Sox-9 proteins, or mutant forms or fusionproteins thereof, etc.).

For example, an expression vector comprises one nucleic acid encoding atranscription factor operably linked to a promoter. In expressionvectors comprising two nucleic acids encoding transcription factors,each nucleic acid may be operably linked to a promoter. The promoteroperably linked to each nucleic acid may be different or the same.Alternatively, the two nucleic acids may be operably linked to a singlepromoter. Promoters useful for the expression vectors disclosed herecould be any promoter known in the art. The ordinarily skilled artisancould readily determine suitable promoters for the host cell in whichthe nucleic acid is to be expressed, the level of expression of proteindesired, or the timing of expression, etc. The promoter may be aconstitutive promoter, an inducible promoter, or a cell-type specificpromoter.

The recombinant expression vectors disclosed here can be designed forexpression of PDX-1 in prokaryotic or eukaryotic cells. For example,PDX-1, Pax-4, MafA, NeuroD1, and/or Sox-9 can be expressed in bacterialcells such as E. coli, insect cells (using baculovirus expressionvectors) yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences disclosedhere can be carried out by standard DNA synthesis techniques.

In another embodiment, the PDX-1, Pax-4, MafA, NeuroD1, or Sox-9expression vector is a yeast expression vector. Examples of vectors forexpression in yeast S. cerevisiae include pYepSec1 (Baldari, et al.,(1987) EMBO J 6:229-234), pMFa (Kujan and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp,San Diego, Calif.).

Alternatively, PDX-1, Pax-4, MafA, NeuroD1 or Sox-9 can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith et al. (1983) Mol Cell Biol3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid disclosed here is expressed inmammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

The disclosure herein, further provides a recombinant expression vectorcomprising a DNA molecule disclosed here cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to PDX mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosen thatdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen that direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al., “Antisense RNA asa molecular tool for genetic analysis,” Reviews—Trends in Genetics, Vol.1(1) 1986.

Another embodiment disclosed herein pertains to host cells into which arecombinant expression vector disclosed here has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. Additionally, hostcells could be modulated once expressing PDX-1, Pax-4, MafA, NeuroD1 orSox-9 or a combination thereof, and may either maintain or looseoriginal characteristics.

A host cell can be any prokaryotic or eukaryotic cell. For example,PDX-1, Pax-4, MafA, NeuroD1 or Sox-9 protein can be expressed inbacterial cells such as E. coli, insect cells, yeast or mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells). Alternatively,a host cell can be a premature mammalian cell, i.e., pluripotent stemcell. A host cell can also be derived from other human tissue. Othersuitable host cells are known to those skilled in the art.

Vector DNA may be introduced into prokaryotic or eukaryotic cells viaconventional transformation, transduction, infection or transfectiontechniques. A skilled artisan would appreciate that the terms“transformation” “transduction”, “infection” and “transfection” mayencompass a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. In addition, transfectioncan be mediated by a transfection agent. A skilled artisan wouldappreciate that the term “transfection agent” may encompass any compoundthat mediates incorporation of DNA in the host cell, e.g., liposome.Suitable methods for transforming or transfecting host cells can befound in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

Transfection may be “stable” (i.e. integration of the foreign DNA intothe host genome) or “transient” (i.e., DNA is episomally expressed inthe host cells) or mRNA is electroporated into cells).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome theremainder of the DNA remains episomal In order to identify and selectthese integrants, a gene that encodes a selectable marker (e.g.,resistance to antibiotics) is generally introduced into the host cellsalong with the gene of interest. Various selectable markers includethose that confer resistance to drugs, such as G418, hygromycin andmethotrexate. Nucleic acid encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding PDX-1 orcan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die). In another embodiment the cells modulated byPDX-1 or the transfected cells are identified by the induction ofexpression of an endogenous reporter gene. In a specific embodiment, thepromoter is the insulin promoter driving the expression of greenfluorescent protein (GFP).

In one embodiment the PDX-1, Pax-4, MafA, NeuroD1, or Sox-9 nucleic acidis present in a viral vector. In one embodiment, the PDX-1 and NeuroD1nucleic acids are present in the same viral vector. In anotherembodiment, the PDX-1 and Pax4 nucleic acids are present in the sameviral vector. In another embodiment the PDX-1, Pax-4, MafA, NeuroD1, orSox-9 nucleic acid is encapsulated in a virus. In another embodiment,the PDX-1 and NeuroD1 is encapsulated in a virus (i.e., nucleic acidsencoding PDX-1 and NeuroD1 are encapsulated in the same virus particle).In another embodiment, the PDX-1 and Pax4 are encapsulated in a virus(i.e., nucleic acids encoding PDX-1 and Pax4 are encapsulated in thesame virus particle). In some embodiments the virus preferably infectspluripotent cells of various tissue types, e.g. hematopoietic stem,cells, neuronal stem cells, hepatic stem cells or embryonic stem cells,preferably the virus is hepatotropic. A skilled artisan would appreciatethat the term “hepatotropic” it is meant that the virus has the capacityto preferably target the cells of the liver either specifically ornon-specifically. In further embodiments the virus is a modulatedhepatitis virus, SV-40, or Epstein-Bar virus. In yet another embodiment,the virus is an adenovirus.

Gene Therapy

In one embodiment, a nucleic acid or nucleic acids encoding a PDX-1,Pax-4, MafA, NeuroD1, or Sox-9 polypeptide or a combination thereof, asdisclosed herein, or functional derivatives thereof, are administered byway of gene therapy. Gene therapy refers to therapy that is performed bythe administration of a specific nucleic acid to a subject. In oneembodiment, the nucleic acid produces its encoded peptide(s), which thenserve to exert a therapeutic effect by modulating function of anaforementioned disease or disorder. e.g., diabetes. Any of themethodologies relating to gene therapy available within the art may beused in the practice of the disclosure presented herein. See e.g.,Goldspiel, et al., 1993. Clin Pharm 12: 488-505.

In another embodiment, the therapeutic comprises a nucleic acid that ispart of an expression vector expressing any one or more of theaforementioned PDX-1, Pax-4, MafA, NeuroD1, and/or Sox-9 polypeptides,or fragments, derivatives or analogs thereof, within a suitable host. Inone embodiment, such a nucleic acid possesses a promoter that isoperably linked to coding region(s) of a PDX-1, Pax-4, MafA, NeuroD1 andSox-9 polypeptide. The promoter may be inducible or constitutive, and,optionally, tissue-specific. The promoter may be, e.g., viral ormammalian in origin. In another specific embodiment, a nucleic acidmolecule is used in which coding sequences (and any other desiredsequences) are flanked by regions that promote homologous recombinationat a desired site within the genome, thus providing forintra-chromosomal expression of nucleic acids. See e.g., Koller andSmithies, 1989. Proc Natl Acad Sci USA 86: 8932-8935. In yet anotherembodiment, the nucleic acid that is delivered remains episomal andinduces an endogenous and otherwise silent gene.

Delivery of the therapeutic nucleic acid into a patient may be eitherdirect (i.e., the patient is directly exposed to the nucleic acid ornucleic acid-containing vector) or indirect (i.e., cells are firstcontacted with the nucleic acid in vitro, then transplanted into thepatient). These two approaches are known, respectively, as in vivo or exvivo gene therapy. In another embodiment, a nucleic acid is directlyadministered in vivo, where it is expressed to produce the encodedproduct. This may be accomplished by any of numerous methods known inthe art including, but not limited to, constructing said nucleic acid aspart of an appropriate nucleic acid expression vector and administeringthe same in a manner such that it becomes intracellular (e.g., byinfection using a defective or attenuated retroviral or other viralvector; see U.S. Pat. No. 4,980,286); directly injecting naked DNA;using microparticle bombardment (e.g., a “Gene Gun®; Biolistic, DuPont);coating said nucleic acids with lipids; using associated cell-surfacereceptors/transfecting agents; encapsulating in liposomes,microparticles, or microcapsules; administering it in linkage to apeptide that is known to enter the nucleus; or by administering it inlinkage to a ligand predisposed to receptor-mediated endocytosis (see,e.g., Wu and Wu, 1987. J Biol Chem 262: 4429-4432), which can be used to“target” cell types that specifically express the receptors of interest,etc.

An additional approach to gene therapy involves transferring a gene ormRNA into cells in in vitro tissue culture by such methods aselectroporation, lipofection, calcium phosphate-mediated transfection,viral infection, or the like. Generally, the methodology of transferincludes the concomitant transfer of a selectable marker to the cells.The cells are then placed under selection pressure (e.g., antibioticresistance) so as to facilitate the isolation of those cells that havetaken up, and are expressing, the transferred gene. Those cells are thendelivered to a patient. In another embodiment, prior to the in vivoadministration of the resulting recombinant cell, the nucleic acid isintroduced into a cell by any method known within the art including, butnot limited to: transfection, electroporation, microinjection, infectionwith a viral or bacteriophage vector containing the nucleic acidsequences of interest, cell fusion, chromosome-mediated gene transfer,microcell-mediated gene transfer, spheroplast fusion, and similarmethodologies that ensure that the necessary developmental andphysiological functions of the recipient cells are not disrupted by thetransfer. See e.g., Loeffler and Behr, 1993. Meth Enzymol 217: 599-618.The chosen technique should provide for the stable transfer of thenucleic acid to the cell, such that the nucleic acid is expressible bythe cell. In yet another embodiment, said transferred nucleic acid isheritable and expressible by the cell progeny. In an alternativeembodiment, the transferred nucleic acid remains episomal and inducesthe expression of the otherwise silent endogenous nucleic acid.

In one embodiment, the resulting recombinant cells may be delivered to apatient by various methods known within the art including, but notlimited to, injection of epithelial cells (e.g., subcutaneously),application of recombinant skin cells as a skin graft onto the patient,and intravenous injection of recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) or liver cells. The total numberof cells that are envisioned for use depend upon the desired effect,patient state, and the like, and may be determined by one skilled withinthe art. In one embodiment, at least 10⁶ transdifferentiated cells areneeded for use in a method of treating as disclosed herein. In anotherembodiment, at least 10⁷ transdifferentiated cells, at least 10⁸transdifferentiated cells, at least 10⁹ transdifferentiated cells, or atleast 10¹⁰ transdifferentiated cells are needed for use in a method oftreating as disclosed herein. In yet another embodiment, about 1.8×10⁹transdifferentiated cells are needed for use in a method of treating asdisclosed herein.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

DNA for gene therapy can be administered to patients parenterally, e.g.,intravenously, subcutaneously, intramuscularly, and intraperitoneally.DNA or an inducing agent is administered in a pharmaceuticallyacceptable carrier, i.e., a biologically compatible vehicle that issuitable for administration to an animal e.g., physiological saline. Atherapeutically effective amount is an amount that is capable ofproducing a medically desirable result, e.g., an increase of apancreatic gene in a treated animal. Such an amount can be determined byone of ordinary skill in the art. As is well known in the medical arts,dosage for any given patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages may vary, but apreferred dosage for intravenous administration of DNA is approximately10⁶ to 10²² copies of the DNA molecule. For example the DNA isadministers at approximately 2×10¹² virions per Kg.

Methods of Manufacturing Human Insulin Producing (IP) Cells

Manufacturing of human insulin producing cells may overcome the shortageof tissue available for cell-based therapies, for instance for treatinga subject suffering from type I Diabetes Mellitus. Methods ofmanufacturing human insulin producing cells in sufficient numbers, inone embodiment, provides a cell-based product for use in these and othertherapies, as disclosed herein (FIG. 32).

Reference is now made to FIG. 34, which presents a flowchart of amanufacturing process of the human insulin producing cell product, whichmay in one embodiment be autologous or allogeneic insulin producingcells (AIP). FIG. 34 describes one embodiment of a manufacturing processof human insulin producing cells, wherein the starting materialcomprises liver tissue. A skilled artisan would recognize that anysource of non-pancreatic β-cell tissue could be used in thismanufacturing process.

Embodiments for many of the steps presented in FIG. 34 are described indetail throughout this application, and will not be repeated herein,though they should be considered herein. Reference is also made toExamples 20 and 21, which exemplify many of these steps. In brief, themanufacturing process may be understood based on the steps presentedbelow.

As indicated at Step 1: Obtaining Liver Tissue. In one embodiment, livertissue is human liver tissue. In another embodiment, the liver tissue isobtained as part of a biopsy. In another embodiment, liver tissue isobtained by way of any surgical procedure known in the art. In anotherembodiment, obtaining liver tissue is performed by a skilled medicalpractitioner. In another embodiment, liver tissue obtained is livertissue from a healthy individual. In a related embodiment, the healthyindividual is an allogeneic donor for a patient in need of a cell-basedtherapy that provides processed insulin in a glucose regulated manner,for example a type I Diabetes mellitus patient or a patient sufferingfor pancreatitis. In another embodiment, donor Screening and DonorTesting was performed to ensure that tissue obtained from donors showsno clinical or physical evidence of or risk factors for infectious ormalignant diseases were from manufacturing of AIP cells. In yet anotherembodiment, liver tissue is obtained from a patient in need of acell-based therapy that provides processed insulin in a glucoseregulated manner, for example a type I Diabetes mellitus patient or apatient suffering for pancreatitis. In still another embodiment, livertissue is autologous with a patient in need of a cell-based therapy thatprovides processed insulin in a glucose regulated manner, for example atype I Diabetes mellitus patient or a patient suffering forpancreatitis.

As indicated at Step 2: Recovery and Processing of Primary Liver Cells.Liver tissue is processed using well know techniques in the art forrecovery of adherent cells to be used in further processing. In oneembodiment, liver tissue is cut into small pieces of about 1-2 mm andgently pipetted up and down in sterile buffer solution. The sample maythen be incubated with collagenase to digest the tissue. Following aseries of wash steps, in another embodiment, primary liver cells may beplated on pre-treated fibronectin-coated tissue culture tissue dishes.The skilled artisan would know well how to then process (passage) thecells following well-known techniques for propagation of liver cells.Briefly, cells may be grown in a propagation media and through a seriesof seeding and harvesting cell number is increased. Cells may be splitupon reaching 80% confluence and re-plated. FIG. 33 (0-2 weeks) shows aschematic of one embodiment of this recovery and process steprepresenting 2 passages of the primary liver cells.

A skilled artisan would appreciate the need for sufficient cells at, forexample the 2 week time period, prior to beginning the expansion phaseof the protocol (step 3). The skilled artisan would recognize that the2-week time period is one example of a starting point for expandingcells, wherein cells may be ready for expansion be before or after thistime period. In one embodiment, recovery and processing of primary cellsyields at least 1×10⁵ cells after two passages of the cells. In anotherembodiment, recovery and processing of primary cells yields at least1×10⁶ cells after two passages of the cells. In another embodiment,recovery and processing of primary cells yields at least 2×10⁶ cellsafter two passages of the cells. In another embodiment, recovery andprocessing of primary cells yields at least 5×10⁶ cells after twopassages of the cells. In another embodiment, recovery and processing ofprimary cells yields at least 1×10⁷ cells after two passages of thecells. In another embodiment, recovery and processing of primary cellsyields between 1×10⁵-1×10⁶ cells after two passages of the cells. Inanother embodiment, recovery and processing of primary cells yieldsbetween 1×10⁶-1×10⁷ cells after two passages of the cells. In anotherembodiment, enough starting tissue is used to ensure the recovery andprocessing of primary cells yields enough cells after two passages foran adequate seeding density at Step 3, large-scale expansion of thecells.

In one embodiment, 1^(st) passage primary cells are cryopreserved forlater use. In another embodiment, early passage primary cells arecryopreserved for later use. In yet another embodiment, 2^(nd) passageprimary cells are cryopreserved for later use.

As indicated at Step 3: Propagation/Proliferation of Primary Liver Cells

Step 3 represents the large-scale expansion phase of the manufacturingprocess. A skilled artisan would appreciate the need for sufficientcells at the 5 week time period, prior to beginning thetransdifferentiation phase of the protocol (step 4), wherein apredetermined number of cells may be envisioned to be needed fortreating a patient. In one embodiment, the predetermined number of cellsneeded prior to transdifferentiation comprises about 1×10⁸ primarycells. In another embodiment, the predetermined number of cells neededprior to transdifferentiation comprises about 2×10⁸ primary cells. Inone embodiment, the predetermined number of cells needed prior totransdifferentiation comprises about 3×10⁸ primary cells, 4×10⁸ primarycells, 5×10⁸ primary cells, 6×10⁸ primary cells, 7×10⁸ primary cells,8×10⁸ primary cells, 9×10⁸ primary cells, 1×10⁹ primary cells, 2×10⁹primary cells, 3×10⁹ primary cells, 4×10⁹ primary cells, 5×10⁹ primarycells, 6×10⁹ primary cells, 7×10⁹ primary cells, 8×10⁹ primary cells,9×10⁹ primary cells, or 1×10¹⁰ primary cells.

In one embodiment, the cell seeding density at the time of expansioncomprises 1×10³-10×10³ cell/cm². In another embodiment, the cell seedingdensity at the time of expansion comprises 1×10³-8×10³ cell/cm². Inanother embodiment, the cell seeding density at the time of expansioncomprises 1×10³-5×10³ cell/cm². In another embodiment, the cell seedingdensity at the time of expansion comprises 1×10³. In another embodiment,the cell seeding density at the time of expansion comprises 2×10³. Inanother embodiment, the cell seeding density at the time of expansioncomprises 3×10³. In another embodiment, the cell seeding density at thetime of expansion comprises 4×10³. In another embodiment, the cellseeding density at the time of expansion comprises 5×10³. In anotherembodiment, the cell seeding density at the time of expansion comprises6×10³. In another embodiment, the cell seeding density at the time ofexpansion comprises 7×10³. In another embodiment, the cell seedingdensity at the time of expansion comprises 8×10³. In another embodiment,the cell seeding density at the time of expansion comprises 9×10³. Inanother embodiment, the cell seeding density at the time of expansioncomprises 10×10³.

In another embodiment, the range for cells seeding viability at the timeof expansion comprises 60-100%. In another embodiment, the range forcells seeding viability at the time of expansion comprises a viabilityof about 70-99%. In another embodiment, the cell seeding viability atthe time of expansion comprises a viability of about 60%. In anotherembodiment, the cell seeding viability at the time of expansioncomprises a viability of about 65%. In another embodiment, the cellseeding viability at the time of expansion comprises a viability ofabout 70%. In another embodiment, the cell seeding viability at the timeof expansion comprises a viability of about 75%. In another embodiment,the cell seeding viability at the time of expansion comprises aviability of about 80%. In another embodiment, the cell seedingviability at the time of expansion comprises a viability of about 85%.In another embodiment, the cell seeding viability at the time ofexpansion comprises a viability of about 90%. In another embodiment, thecell seeding viability at the time of expansion comprises a viability ofabout 95%. In another embodiment, the cell seeding viability at the timeof expansion comprises a viability of about 99%. In another embodiment,the cell seeding viability at the time of expansion comprises aviability of about 99.9%.

FIG. 33 schematically illustrates one embodiment of this expansionperiod. In one embodiment, expansion occurs between weeks 2 and 5. Theskilled artisan would recognize variability within starting tissuematerial (FIG. 29). Therefore, in another embodiment expansion occursbetween weeks 2 and 6. In still another embodiment, expansion occursbetween weeks 2 and 7. In another embodiment, expansion occurs betweenweeks 2 and 4. In yet another embodiment, expansion occurs until theneeded number of primary cells has been propagated. For example, FIG. 28shows that a target goal of 1 billion cells was reached by day 30 ofculture.

A skilled artisan would appreciate that concurrent with expansion ofcells, the population could be enhanced for transdifferentiation.Description of primary adult liver cells enhanced fortransdifferentiation and methods for enriching these populations havebeen disclosed herein, and are exemplified in Examples 10-17 and 23. Inone embodiment, selection for GSRE activity is used to enrich apopulation of adult cells for transdifferentiation. In anotherembodiment, levels of gene expression are measured for genes known tohave either increased or decreased expression, wherein such increases ordecreases indicate predisposition to transdifferentiation. In anotherembodiment, primary adult liver cells may be incubated with lithiumprior to transdifferentiation, wherein the incubation enhancespredisposition of a population of cells within said population ofprimary adult liver cells.

In one embodiment, bioreactors are used to expand and propagate primarycells prior to the transdifferentiation step. Bioreactors may be used orcultivation of cells, in which conditions are suitable for high cellconcentrations (See Example 20). In another embodiment, a bioreactorprovides a closed system for expansion of cells. In another embodiment,multiple bioreactors are used in a series for cell expansion. In anotherembodiment, a bioreactor used in the methods disclosed herein is asingle use bioreactor. In another embodiment, a bioreactor used is amulti-use bioreactor. In yet another embodiment, a bioreactor comprisesa control unit for monitoring and controlling parameters of the process.In another embodiment, parameters for monitoring and controllingcomprise Dissolve Oxygen (DO), pH, gases, and temperature.

As indicated at Step 4: Transdifferentiation (TD) of primary LiverCells.

In one embodiment, transdifferentiation comprises any method oftransdifferentiation disclosed herein. For example, transdifferentiationmay comprise a hierarchy (1+1+1) protocol or a “2+1” protocol, asdisclosed herein.

In one embodiment, the resultant cell population followingtransdifferentiation comprises transdifferentiated cells having apancreatic phenotype and function. In another embodiment, the resultantcell population following transdifferentiation comprisestransdifferentiated cells having a mature β-cell pancreatic phenotypeand function. In another embodiment, the resultant cell populationfollowing transdifferentiation comprises transdifferentiated cellshaving increased insulin content. In another embodiment, the resultantcell population following transdifferentiation comprisestransdifferentiated cells able to secrete processed insulin in aglucose-regulated manner. In another embodiment, the resultant cellpopulation following transdifferentiation comprises transdifferentiatedcells has increased C-peptide levels.

In another embodiment, the resultant cell population followingtransdifferentiation comprises transdifferentiated cells havingincreased endogenous expression of at least one pancreatic gene marker.In another embodiment, endogenous expression is increased for at leasttwo pancreatic gene markers. In another embodiment, endogenousexpression is increased for at least three pancreatic gene markers. Inanother embodiment, endogenous expression is increased for at least fourpancreatic gene markers. In a related embodiment, pancreatic genemarkers comprise PDX-1, NeuroD1, MafA, Nkx6.1, glucagon, somatostatinand Pax4.

In one embodiment, endogenous PDX-1 expression is greater than 10² foldover non-differentiated cells. In another embodiment, endogenous PDX-1expression is greater than 10³ fold over non-differentiated cells. Inanother embodiment, endogenous PDX-1 expression is greater than 10⁴ foldover non-differentiated cells. In another embodiment, endogenous PDX-1expression is greater than 10⁵ fold over non-differentiated cells. Inanother embodiment, endogenous PDX-1 expression is greater than 10⁶ foldover non-differentiated cells.

In another embodiment, endogenous NeuroD1 expression is greater than 10²fold over non-differentiated cells. In another embodiment, endogenousNeuroD1 expression is greater than 10³ fold over non-differentiatedcells. In another embodiment, endogenous NeuroD1 expression is greaterthan 10⁴ fold over non-differentiated cells. In another embodiment,endogenous NeuroD1 expression is greater than 10⁵ fold overnon-differentiated cells.

In another embodiment, endogenous MafA expression is greater than 10²fold over non-differentiated cells. In another embodiment, endogenousMafA expression is greater than 10³ fold over non-differentiated cells.In another embodiment, endogenous MafA expression is greater than 10⁴fold over non-differentiated cells. In another embodiment, endogenousMafA expression is greater than 10⁵ fold over non-differentiated cells.

In another embodiment, endogenous glucagon expression is greater than 10fold over non-differentiated cells. In another embodiment, endogenousglucagon expression is greater than 10² fold over non-differentiatedcells. In another embodiment, endogenous glucagon expression is greaterthan 10³ fold over non-differentiated cells.

In another embodiment, endogenous expression of PDX-1, NeuroD1, or MafA,or any combination thereof is each greater than 60% overnon-differentiated cells. In another embodiment, endogenous expressionof PDX-1, NeuroD1, or MafA, or any combination thereof is each greaterthan 70% over non-differentiated cells. In another embodiment,endogenous expression of PDX-1, NeuroD1, or MafA, or any combinationthereof is each greater than 80% over non-differentiated cells

In another embodiment, the resultant cell population followingtransdifferentiation comprises transdifferentiated cells having at least60% viability. In another embodiment, the resultant cell populationfollowing transdifferentiation comprises transdifferentiated cellshaving at least 70% viability. In another embodiment, the resultant cellpopulation following transdifferentiation comprises transdifferentiatedcells having at least 80% viability. In another embodiment, theresultant cell population following transdifferentiation comprisestransdifferentiated cells having at least 90% viability.

In another embodiment, the resultant cell population followingtransdifferentiation comprises transdifferentiated cells showingdecreased liver cell markers. In another embodiment, the resultant cellpopulation following transdifferentiation comprises transdifferentiatedcells showing decreased albumin or alpha-1 antitrypsin (AAT), or anycombination. In another embodiment, the resultant cell populationfollowing transdifferentiation comprises transdifferentiated cellscomprising less than 1% by FACS albumin or alpha-1 antitrypsin (AAT), orany combination.

In another embodiment, transdifferentiated cells maintain a pancreaticphenotype and function for at least 6 months. In another embodiment,transdifferentiated cells maintain a pancreatic phenotype and functionfor at least 12 months. In another embodiment, transdifferentiated cellsmaintain a pancreatic phenotype and function for at least 18 months. Inanother embodiment, transdifferentiated cells maintain a pancreaticphenotype and function for at least 24 months. In another embodiment,transdifferentiated cells maintain a pancreatic phenotype and functionfor at least 36 months. In another embodiment, transdifferentiated cellsmaintain a pancreatic phenotype and function for at least 48 months. Inanother embodiment, transdifferentiated cells maintain a pancreaticphenotype and function for at least 4 years. In another embodiment,transdifferentiated cells maintain a pancreatic phenotype and functionfor at least 5 years.

In one embodiment, cell number is maintained duringtransdifferentiation. In another embodiment, cell number decreases byless than 5% during transdifferentiation. In another embodiment, cellnumber decreases by less than 10% during transdifferentiation. Inanother embodiment, cell number decreases by less than 15% duringtransdifferentiation. In another embodiment, cell number decreases byless than 20% during transdifferentiation. In another embodiment, cellnumber decreases by less than 25% during transdifferentiation.

As indicated at Step 5: Harvest Transdifferentiated Primary Liver Cells

In one embodiment, transdifferentiated primary liver cells comprisinghuman insulin producing cells are harvested and used for a cell-basedtherapy. In one embodiment, cell number is maintained during harvesting.In another embodiment, cell number decreases by less than 5% duringharvesting. In another embodiment, cell number decreases by less than10% during harvesting. In another embodiment, cell number decreases byless than 15% during harvesting. In another embodiment, cell numberdecreases by less than 20% during harvesting. In another embodiment,cell number decreases by less than 25% during harvesting.

In one embodiment, the number of transdifferentiated cells recovered atharvest is about 1×10-1×10¹⁰ cells total. In another embodiment, thenumber of transdifferentiated cells recovered at harvest is about1×10⁸-1×10¹⁰ cells total. In another embodiment, the number oftransdifferentiated cells recovered at harvest is about 1×10⁷-1×10⁹cells total. In another embodiment, the number of transdifferentiatedcells recovered at harvest is about 1×10⁷ cells total. In anotherembodiment, the number of transdifferentiated cells recovered at harvestis about 5×10⁷ cells total. In another embodiment, the number oftransdifferentiated cells recovered at harvest is about 7.5×10⁷ cellstotal. In another embodiment, the number of transdifferentiated cellsrecovered at harvest is about 1×10⁸ cells total. In another embodiment,the number of transdifferentiated cells recovered at harvest is about2.5×10⁸ cells total. In another embodiment, the number oftransdifferentiated cells recovered at harvest is about 5×10⁸ cellstotal. In another embodiment, the number of transdifferentiated cellsrecovered at harvest is about 7.5×10⁸ cells total. In anotherembodiment, the number of transdifferentiated cells recovered at harvestis about 1×10⁹ cells total. In another embodiment, the number oftransdifferentiated cells recovered at harvest is about 2×10⁸ cellstotal. In another embodiment, the number of transdifferentiated cellsrecovered at harvest is about 3×10⁸ cells total. In another embodiment,the number of transdifferentiated cells recovered at harvest is about4×10⁹ cells total. In another embodiment, the number oftransdifferentiated cells recovered at harvest is about 5×10⁹ cellstotal. In another embodiment, the number of transdifferentiated cellsrecovered at harvest is about 6×10⁹ cells total. In another embodiment,the number of transdifferentiated cells recovered at harvest is about7×10⁹ cells total. In another embodiment, the number oftransdifferentiated cells recovered at harvest is about 8×10⁹ cellstotal. In another embodiment, the number of transdifferentiated cellsrecovered at harvest is about 9×10⁹ cells total.

In one embodiment, the density of transdifferentiated cells at harvestis about 1×10³-1×10⁵ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 1×10⁴-5×10⁴ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 1×10⁴-4×10⁴ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 1×10³ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 2×10³ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 3×10³ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 4×10³ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 5×10³ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 6×10³ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 7×10³ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 8×10³ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 9×10³ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 1×10⁴ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 2×10⁴ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 3×10⁴ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 4×10⁴ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 5×10⁴ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 6×10⁴ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 7×10⁴ cells/cm². In another embodiment, the density oftransdifferentiated cells at harvest is about 8×10⁴ cells/cm². Inanother embodiment, the density of transdifferentiated cells at harvestis about 9×10⁴ cells/cm².

In another embodiment, the range for cell viability at the time ofharvesting comprises 50-100%. In another embodiment, the range for cellviability at the time of harvesting comprises 60-100%. In anotherembodiment, the range for cell viability at the time of harvestingcomprises 50-90%. In another embodiment, the range for cell viability atthe time of harvesting comprises a viability of about 60-99%. In anotherembodiment, the range for cell viability at the time of harvestingcomprises a viability of about 60-90%. In another embodiment, the cellviability at the time of harvesting comprises a viability of about 60%.In another embodiment, the cell viability at the time of harvestingcomprises a viability of about 65%. In another embodiment, the cellviability at the time of harvesting comprises a viability of about 70%.In another embodiment, the cell viability at the time of harvestingcomprises a viability of about 75%. In another embodiment, the cellviability at the time of harvesting comprises a viability of about 80%.In another embodiment, the cell viability at the time of harvestingcomprises a viability of about 85%. In another embodiment, the cellviability at the time of harvesting comprises a viability of about 90%.In another embodiment, the cell viability at the time of harvestingcomprises a viability of about 95%. In another embodiment, the cellviability at the time of harvesting comprises a viability of about 99%.In another embodiment, the cell viability at the time of harvestingcomprises a viability of about 99.9%.

In another embodiment, transdifferentiated primary liver cellscomprising human insulin producing cells are harvested and stored foruse in a cell-based therapy at a later date. In another embodiment,storage comprises cryopreserving the cells.

As indicated at Step 6: Quality Analysis/Quality Control

Before any use of transdifferentiated cells in a cell-based therapy, thetransdifferentiated cells must undergo a quality analysis/qualitycontrol assessment. FACS analysis and/or RT-PCR may be used toaccurately determine membrane markers and gene expression. Further,analytical methodologies for insulin secretion are well known in the artincluding ELISA, MSD, ELISpot, HPLC, RP-HPLC. In one embodiment, insulinsecretion testing is at low glucose concentrations (about 2 mM) incomparison to high glucose concentrations (about 17.5 mM).

Therapeutics Compositions

The herein-described transdifferentiation-inducing compounds, or ectopicpancreatic transcription factors (i.e., PDX-1, Pax-4, MafA, NeuroD1 orSox-9 polypeptides, ribonucleic acids or nucleic acids encoding PDX-1,Pax-4, MafA, NeuroD1 or Sox-9 polypeptides) and the cells having apancreatic beta cell phenotype produced by the methods disclosed here,when used therapeutically, are referred to herein as “Therapeutics”.Methods of administration of Therapeutics include, but are not limitedto, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The Therapeutics ofthe disclosure presented herein may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically-active agents. Administration can be systemic or local,e.g. through portal vein delivery to the liver. In addition, it may beadvantageous to administer the Therapeutic into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection. Intraventricular injection may be facilitated by anintraventricular catheter attached to a reservoir (e.g., an Ommayareservoir). Pulmonary administration may also be employed by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. It mayalso be desirable to administer the Therapeutic locally to the area inneed of treatment; this may be achieved by, for example, and not by wayof limitation, local infusion during surgery, topical application, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant. Various delivery systems are known and can be usedto administer a Therapeutic of the disclosure presented hereinincluding, e.g.: (i) encapsulation in liposomes, microparticles,microcapsules; (ii) recombinant cells capable of expressing theTherapeutic; (iii) receptor-mediated endocytosis (See, e.g., Wu and Wu,1987. J Biol Chem 262:4429-4432); (iv) construction of a Therapeuticnucleic acid as part of a retroviral, adenoviral or other vector, andthe like. In one embodiment of the disclosure presented herein, theTherapeutic may be delivered in a vesicle, in particular a liposome. Ina liposome, the protein of the disclosure presented herein is combined,in addition to other pharmaceutically acceptable carriers, withamphipathic agents such as lipids that exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. No. 4,837,028; andU.S. Pat. No. 4,737,323, all of which are incorporated herein byreference. In yet another embodiment, the Therapeutic can be deliveredin a controlled release system including, e.g.: a delivery pump (See,e.g., Saudek, et al., 1989. New Engl J Med 321:574 and a semi-permeablepolymeric material (See, e.g., Howard, et al., 1989. J Neurosurg71:105). Additionally, the controlled release system can be placed inproximity of the therapeutic target (e.g., the brain), thus requiringonly a fraction of the systemic dose. See, e.g., Goodson, In: MedicalApplications of Controlled Release 1984. (CRC Press, Boca Raton, Fla.).

In one embodiment of the disclosure presented herein, where theTherapeutic is a nucleic acid encoding a protein, the Therapeuticnucleic acid may be administered in vivo to promote expression of itsencoded protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular (e.g., by use of a retroviral vector, by direct injection,by use of microparticle bombardment, by coating with lipids orcell-surface receptors or transfecting agents, or by administering it inlinkage to a homeobox-like peptide which is known to enter the nucleus(See, e.g., Joliot, et al., 1991. Proc Natl Acad Sci USA 88:1864-1868),and the like. Alternatively, a nucleic acid Therapeutic can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination or remain episomal.

In one embodiment, the Therapeutic is a cell having pancreatic beta cellphenotype produced by the methods disclosed here and, the Therapeutic isadministered intravenously. Specifically, the Therapeutic can bedelivered via a portal vein infusion.

A skilled artisan would appreciate that the term “therapeuticallyeffective amount” may encompass total amount of each active component ofthe pharmaceutical composition or method that is sufficient to show ameaningful patient benefit, i.e., treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions.When applied to an individual active ingredient, administered alone, theterm refers to that ingredient alone. When applied to a combination, theterm refers to combined amounts of the active ingredients that result inthe therapeutic effect, whether administered in combination, serially orsimultaneously.

Suitable dosage ranges for intravenous administration of theTherapeutics of the disclosure presented herein are generally at least 1million transdifferentiated cells, at least 2 milliontransdifferentiated cells, at least 5 million transdifferentiated cells,at least 10 million transdifferentiated cells, at least 25 milliontransdifferentiated cells, at least 50 million transdifferentiatedcells, at least 100 million transdifferentiated cells, at least 200million transdifferentiated cells, at least 300 milliontransdifferentiated cells, at least 400 million transdifferentiatedcells, at least 500 million transdifferentiated cells, at least 600million transdifferentiated cells, at least 700 milliontransdifferentiated cells, at least 800 million transdifferentiatedcells, at least 900 million transdifferentiated cells, at least 1billion transdifferentiated cells, at least 2 billiontransdifferentiated cells, at least 3 billion transdifferentiated cells,at least 4 billion transdifferentiated cells, or at least 5 billiontransdifferentiated cells. In one embodiment, the dose is 1-2 billiontransdifferentiated cells into a 60-75 kg subject. One skilled in theart would appreciate that effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.In another embodiment, the effective dose may be administeredintravenously into the liver portal vein.

Cells may also be cultured ex vivo in the presence of therapeuticagents, nucleic acids, or proteins of the disclosure presented herein inorder to proliferate or to produce a desired effect on or activity insuch cells. Treated cells can then be introduced in vivo via theadministration routes described herein for therapeutic purposes.

Pharmaceutical Compositions

The compounds, e.g., PDX-1, Pax-4, MafA, NeuroD1, or Sox-9 polypeptides,nucleic acids encoding PDX-1, Pax-4, MafA, NeuroD1, or Sox-9polypeptides, or a nucleic acid or compound that increases expression ofa nucleic acid that encodes PDX-1, Pax-4, MafA, NeuroD1, or Sox-9polypeptides (also referred to herein as “active compounds”) andderivatives, fragments, analogs and homologs thereof and pancreatic betacells produced by the methods disclosed here, can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, or protein,and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition disclosed here is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms disclosed here are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The nucleic acid molecules disclosed here can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

It should be understood that the disclosure presented herein is notlimited to the particular methodologies, protocols and reagents, andexamples described herein. The terminology and examples used herein isfor the purpose of describing particular embodiments only, for theintent and purpose of providing guidance to the skilled artisan, and isnot intended to limit the scope of the disclosure presented herein.

EXAMPLES Example 1 General Methods

Human Liver Cells

Adult human liver tissues were obtained from individuals 3-23 years oldor older. Liver tissues were used with the approval from the Committeeon Clinical Investigations (the institutional review board). Theisolation of human liver cells was performed as described (Sapir et al,(2005) Proc Natl Acad Sci USA 102: 7964-7969; Meivar-Levy et al, (2007)Hepatology 46: 898-905). The cells were cultured in Dulbecco's minimalessential medium (1 g/l of glucose) supplemented with 10% fetal calfserum, 100 units/ml penicillin; 100 ng/ml streptomycin; 250 ng/mlamphotericin B (Biological Industries, Beit Haemek, Israel), and kept at37° C. in a humidified atmosphere of 5% CO₂ and 95% air.

Viral Infection

The adenoviruses used in this study were as follows: Ad-CMV-Pdx-1 (Sapiret al, 2005 ibid; Meivar-Levy et al, 2007 ibid), Ad-RIP-luciferase(Seijffers et al, (1999) Endocrinology 140: 3311-3317), Ad-CMV-β-Gal,Ad-CMV-MafA (generous gift from Newgard, C. B., Duke University),Ad-CMV-Pax4-IRES-GFP (generous gift from St Onge, L. DeveloGen AG,Göttingen, Germany), and Ad-CMV-Isl1 (generous gift from Kieffer, T.University of British Columbia, Vancouver, Canada). The viral particleswere generated by the standard protocol (He et al, (1998) Proc Natl AcadSci USA 95: 2509-2514).

Liver cells were infected with recombinant adenoviruses for 5-6 days(Table 1) supplemented with EGF (20 ng/ml; Cytolab, Ltd., Israel) andnicotinamide (10 mM; Sigma). The optimal multiplicity of infection (MOI)was determined according to cell survival (<75%) and induction ofC-peptide secretion. The MOI of the viruses used were; Ad-CMV-Pdx-1(1000 MOI), Ad-CMV-Pax4-IRES-GFP (100 MOI), Ad-CMV-MafA (10 MOI) andAd-CMV-Isl1 (100 MOI).

RNA Isolation, RT and RT-PCR Reactions

Total RNA was isolated and cDNA was prepared and amplified, as describedpreviously (Ber et al, (2003) J Biol Chem 278: 31950-31957; Sapir et al,(2005) ibid). Quantitative real-time RT-PCR was performed using ABI Stepone plus sequence Detection system (Applied Biosystems, CA, USA), asdescribed previously (Sapir et al, (2005) ibid; Meivar-Levy et al,(2007) ibid; Aviv et al, (2009) J Biol Chem 284: 33509-33520).

C-Peptide and Insulin Secretion Detection

C-peptide and insulin secretion were measured by static incubations ofprimary cultures of adult liver cells 6 days after the initial exposureto the viral treatment, as described (Sapir et al, (2005) ibid;Meivar-Levy et al, (2007) ibid; Aviv et al, (2009) ibid). Theglucose-regulated C-peptide secretion was measured at 2 mM and 17.5 mMglucose, which was determined by dose-dependent analyses to maximallyinduce insulin secretion from transdifferentiated liver cells, withouthaving adverse effects on treated cells function (Sapir et al, (2005)ibid; Meivar-Levy et al, (2007) ibid; Aviv et al, (2009) ibid).C-peptide secretion was detected by radioimmunoassay using the humanC-peptide radioimmunoassay kit (Linco Research, St. Charles, Mo.; <4%cross-reactivity to human proinsulin). Insulin secretion was detected byradioimmunoassay using human insulin radioimmunoassay kit (DPC, Angeles,Calif.; 32% cross-reactivity to human proinsulin). The secretion wasnormalized to the total cellular protein measured by a Bio-Rad proteinassay kit.

Luciferase Assay

Human liver cells were co-infected with Ad-RIP-luciferase (200 moi) andthe various adenoviruses (as described below). Six days later,luciferase activity was measured using the Luciferase assay System(Promega) and the LKB 1250 Luminometer (LKB, Finland). The results werenormalized to total cellular protein measured by the Bio-Rad ProteinAssay kit (Bio-Rad).

Immunofluorescence

Human liver cells treated with the various adenoviruses, were plated onglass cover slides in 12-well culture plates (2×10⁵ cells/well). 3-4days later, the cells were fixed and stained as described (Sapir et al,(2005) ibid; Meivar-Levy et al, (2007) ibid; Aviv et al, (2009) ibid).The antibodies used in this study were: anti-rabbit PDX-1, anti-goatPDX-1 (both 1:1000 a generous gift from C. V. E. Wright), anti-humaninsulin, anti-human somatostatin (both 1:100, Dako, Glostrup, Denmark),anti-Pax4 (1:100; R&D Systems, Minneapolis, Minn.), anti-MafA (1:160;Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.). The secondaryantibodies used were: anti-rabbit IgG Cyanine (cy2) conjugated antibody1:250, anti-rabbit IgG indocarbocyanine (cy3) conjugated antibody 1:250,anti-goat IgG Cyanine (cy2) conjugated antibody 1:200, anti-goat IgGindocarbocyanine (cy3) conjugated antibody 1:250, and anti-mouse IgGindocarbocyanine (cy3) conjugated antibody 1:250 (all from JacksonImmunoResearch, PA). Finally, the cells were stained with 4′,6-diamidino-2-phenyl-indole (DAPI, Sigma). The slides were imaged andanalyzed using a fluorescent microscope (Provis, Olympus).

Purity Assays

A flow cytometry based assay has been developed as the principal purityassay to ensure that more than 90% of the cells during expansion andtransdifferentiation have a mesenchymal stem cell (MSC) like phenotype.Cultivated MSCs should stain positive for CD73, CD90, CD105 and CD44 andshould be negative for CD45, CD34, CD14 or CD11b, CD19 or CD79α, andHLA-DR surface molecules.

As shown in FIGS. 44A and 44B, expanded liver cells and infected cellsexpressed CD90, CD44, CD105 and CD73 markers at high levels (>90%) whilethey did not express lineage negative markers (cocktail of CD34, CD11b,CD45, CD19 and HLA-DR). To note, CD105 expression was slightly decreasedin infected cells at P16 compared to non-infected cells at P14.Additional experiments are needed to understand if this decrease issignificant and if it decreases with passage numbers or withtransdifferentiation. These results demonstrate that MSC markers werestable over time and during transdifferentiation of liver cells. Flowcytometry for MSC markers may be indeed used as a QC test.

Next step will be to develop flow cytometry or Immunofluorescence assaysto quantify the subpopulations expressing or co-expressing the variousexogenous transcription factors and ideally insulin or C-peptide.

Statistical Analysis

Statistical analyses were performed with a 2-sample Student t-testassuming unequal variances.

Example 2 Pdx-1-Induced Transdifferentiation

Previous studies (Sapir et al, (2005) ibid; Meivar-Levy et al, (2007)ibid; Aviv et al, (2009) ibid; Gefen-Halevi et al, (2010) Cell Reprogram12: 655-664; Meivar-Levy et al, (2011) J Transplant 2011: 252387) havesuggested that PDX-1 alone is capable of inducing β-cell like phenotypeand function in human liver cells, possibly due to its capacity toactivate numerous otherwise silent endogenous pTFs in liver. Theactivation of the pancreatic lineage was fast and occurred within 5 days(Sapir et al, (2005) ibid, Ber et al, (2003) ibid)

In this example, the sequence of events that mediate PDX-1 induced liverto pancreas transdifferentiation is examined. Adenoviral vectorsencoding Pdx-1 were introduced to adult human liver cells, and theeffects of ectopic PDX-1 expression were monitored for four consecutivedays post infection (Days 2-5; FIGS. 1A-1D). Pancreatic hormone andpancreas-specific transcription factor expression was determined byquantitative RT-PCR analysis every day for 5 days. Results werenormalized to β-actin gene expression within the same cDNA sample andare presented as the mean±SE of the relative expression versus controlvirus (Ad-CMV-β-gal, 1000 MOI) treated cells on the same day. Twoindependent experiments were performed, with n≧4, *p<0.05 and **p<0.01.

Both glucagon and somatostatin genes were immediately activated, withinone day after Ad-Pdx-1 infection (FIGS. 1B and 1C). However, insulinexpression was only detected on the fourth to fifth day post-infection(FIG. 1A). To provide a mechanistic explanation for the distincttemporal activation of the three major pancreatic hormones, expressionlevels of endogenously activated transcription factors were analyzedduring the transdifferentiation process. The early pancreatic endocrinetranscription factors, NGN3 and NEUROD1 were immediately activated (FIG.1D). However, β-cell specific TFs, such as NKX6.1 and MafA, were onlygradually and modestly activated in response to ectopic PDX-1expression, reaching their peak expression level on the fourth and fifthday, respectively. The activation of insulin gene expression on thefifth day was associated not only with an increase in MafA expressionbut also with a decrease in Isl1 expression (FIG. 1D). These datasuggest that transdifferentiation of human liver cells along thepancreatic lineage, despite being rapid, is a gradual and consecutiveprocess. The distinct temporal activation of pancreatic hormone geneexpression (such as somatostatin and glucagon) can be partiallyattributed to the time course and the relative levels of theendogenously activated pTFs expression.

Example 3 Combined Expression of Pdx-1, Pax4 and MAFA Increases theEfficiency of Liver to Pancreas Transdifferentiation

Previous studies have suggested that the concerted expression of severalpTFs increases the transdifferentiation efficiency along the β-celllineage, compared to that induced by individual pTFs (Kaneto et al,(2005) Diabetes 54: 1009-1022; Tang et al, (2006) Lab Invest. 86:829-841; Song et al, (2007) Biochem Biophys Res Commun 354: 334-339;Wang et al, (2007) Mol Ther 15: 255-263; Gefen-Halevi et al, (2010)ibid), as well as along other lineages. In order to analyze this notionin the experimental system of primary adult human liver cells describedherein, the individual and joint contribution of three major pTFs onliver to pancreas transdifferentiation were investigated. PDX-1, Pax4and MafA, which mediate different stages in pancreas organogenesis, wereectopically co-expressed in primary cultures of adult human liver cellsusing recombinant adenoviruses. Cultured adult human liver cells wereinfected with Ad-CMV-Pdx-1 (1000 MOI), Ad-CMV-Pax-4 (100 MOI) andAd-CMV-MafA (10 MOI) alone or in concert or with control virus(Ad-CMV-β-gal, 1000 MOI), and pancreatic differentiation markers wereexamined six days later. The multiplicity of infection (MOI) of eachfactor was titrated to result in maximal reprogramming efficiencyassociated by minimal adverse effects on infected cell viability. PDX-1was expressed in 70% of the cells in culture, and the jointco-expression of all 3 pTFs was evident in 46.8% of the PDX-1 positivecells (FIG. 2A). Very few cells stained positive for only Pax-4 or forMafA. Cells that stained positive for all three pTFs are indicated bythe arrows (FIG. 2A, right panel). In FIG. 2B, liver cells wereco-infected with the combined pTFs and with Ad-RIP-LUC (200 moi), andLuciferase activity of the insulin promoter was measured.

The combined expression of the three pTFs resulted in a substantialincrease in insulin promoter activation (FIG. 2B), a three-fold increasein the number of (pro)insulin producing cells (FIG. 2C) and 30-60%increase in glucose regulated (pro)insulin secretion (FIG. 2D), comparedto that induced by each of the pTFs alone. Taken together, these resultssuggest that the combination of the 3 pTFs increase transdifferentiationefficiency and also indicate that each of the factors is limited in itscapacity or is insufficient to individually promote maximaltransdifferentiation (Kaneto et al, (2005) ibid; Tang et al, (2006)ibid; Zhou et al, (2008) Nature 455: 627-632).

Example 4 Maturation and Segregation into the Different HormonesProducing Cells of Transdifferentiated Cells is Temporally Controlled inan Hierarchical Manner

In this example, the impact of temporally controlling the ectopic pTFsexpression was investigated to determine whether increasedtransdifferentiation efficiency by combined ectopic expression of thethree pTFs is also temporally controlled as suggested above (FIGS.2A-2D). In support of temporal control having a role in pancreastransdifferentiation, the three pTFs Pdx-1, Pax4, and MafA displaydistinct temporal expression and function during pancreas organogenesis.

The three pTFs PDX-1, Pax4, and MafA were introduced sequentially or inconcert to primary cultures of adult human liver cells using recombinantadenoviruses. Adenovirus-mediated ectopic gene expression peaks 17 hourspost infection (Varda-Bloom et al, (2001) Gene Ther 8: 819-827).Therefore, the pTFs were sequentially administered during threeconsecutive days (see Viral infection in Example 1), allowing themanifestation of their individual effects. Cells were infected accordingto the schedule as displayed in Table 1.

TABLE 1 Treat- ment Day Day Day Day Day Day order 1 2 3 4 5 6 A Ad-β-galHarvest (control) B Ad-Pdx-1 + Harvest Ad-Pax4 + Ad-MafA C Ad-Pdx-1Ad-Pax4 Ad-MafA Harvest D Ad-MafA Ad-Pax4 Ad-Pdx-1 Harvest E Ad-Pdx1Ad-MafA Ad-Pax4 Harvest

Cells were sequentially infected with one pTF adenoviral construct perday over three days in three different sequences: a direct hierarchicalorder (treatment C=Pdx-1→Pax4→MafA), in an opposite order (treatmentD=MafA→Pax4→Pdx-1), and in a random order (treatment E=Pdx-1→MafA→Pax4).The effect of the sequential pTFs administration on transdifferentiationefficiency and on the β-cell-like maturation was compared to that of theconcerted or simultaneous administration of all three pTFs on the firstday (treatment B=Pdx-1+Pax4+MafA) and to similar MOI of control virus(treatment A=β-gal) (Table 1 and FIG. 3A). Specifically, cultured adulthuman liver cells were infected with Ad-CMV-Pdx-1 (1000 MOI),Ad-CMV-Pax-4 (100 MOI) and Ad-CMV-MafA (10 MOI) together or in asequential manner as summarized in FIG. 3A and Table 1 (treatments B-E)or with control virus (Ad-CMV-β-gal, 1000 moi, treatment A), andanalyzed for their pancreatic differentiation six days later.

Insulin promoter activity (FIG. 4A), the percent of insulin producingcells (FIG. 3B) and glucose-regulated (pro)insulin secretion (FIG. 3C)were unaffected by the order of the sequentially administered pTFs.Interestingly, the sequential pTF administration in the random order(treatment E=Pdx-1→MafA→Pax4) resulted in increased insulin promoteractivity but was associated with loss of glucose regulation of insulinsecretion and decreased glucose transporter 2 (GLUT-2) expression (FIGS.3B, 3C and 4B). Loss of glucose sensing ability upon changing the orderof Pax4 and MafA administration suggests a potential effect of thesequence of expressed pTFs on β-cell-like maturation but not on theefficiency of the transdifferentiation process.

Example 5 Hierarchical Administration of Pdx-1, Pax4, and MAFA Promotesthe Maturation of Transdifferentiated Cells to β-Like Cells

The previous results encouraged further investigation to determine towhat extent and under which conditions increased transdifferentiationefficiency is associated with enhanced maturation along the β-celllineage. The hallmark characteristics of mature β-cells are the capacityto process the proinsulin and secrete it in a glucose-regulated manner(Eberhard et al, (2009) Curr Opin Genet Dev 19: 469-475; Borowiak,(2010) Rev Diabet Stud 7: 93-104). To analyze whether the temporalchanges in pTF expression distinctly affect transdifferentiated cellmaturation along the β-cell lineage, the effect of the distincttreatments A-E (Table 1 and FIG. 3A) on proinsulin processing andglucose-regulated c-peptide secretion was analyzed.

Indeed, only the direct hierarchical administration (treatment C) of thepTFs resulted in pronounced production of processed insulin and itsglucose-regulated secretion that displayed physiological glucose doseresponse characteristics (FIGS. 3C and 5A). The newly acquired phenotypeand function were stable, as demonstrated by the ability to secretec-peptide in a glucose-regulated manner for up to four weeks in vitro(FIGS. 5A and 5B).

The increased prohormone processing only upon the direct hierarchicalpTFs administration (treatment C) was associated with pronouncedincrease in PCSK2 and GLUT2 gene expression, which possess roles inprohormone processing and glucose sensing abilities, respectively (FIGS.3A-3E and 4A-4C). These data suggest an obligatory role for thesequential and direct hierarchical expression of pTFs in promoting thematuration and function of the transdifferentiated liver cells along theβ-cell lineage. Both concerted (treatment B) and sequential TFadministration in an indirect hierarchical mode (treatment D and E),failed to generate transdifferentiated cells that display matureβ-cell-like characteristics.

To provide a mechanistic explanation for the changes in the β-cell-likestate of maturation the repertoire of the endogenously activated pTFsunder the distinct temporal treatments (B-E) was analyzed. All thetreatments (B-E) resulted in increased expression of numerous endogenouspTFs (FIG. 3E), such as NEUROG3, NEUROD1, NKX6.1 and NKX2.2. However,the most robust difference between the “mature” (treatment C) and“immature” phenotypes (treatments B, E and D) was exhibited at thelevels of the endogenous Isl1 gene expression. Thus, the most enhancedmaturation along the β-cell lineage induced by direct hierarchical pTFsadministration (treatment C) correlates with a dramatic decrease inendogenous Isl1 expression (FIG. 3E, arrow). Taken together these datasuggest that the maturation of transdifferentiated cells to β cells maybe affected by the relative and temporal expression levels of specificpTFs.

Example 6 Hierarchical Administration of Pdx-1, Pax4, and MAFA Promotesthe Segregation of Transdifferentiated Cells Between β-Like and δ-LikeCells

Exclusion of MafA from treatment C (Table 1) induced both Isl-1 (FIG.6D) and somatostatin gene expression (FIG. 8D). To analyze whether Isl-1increased expression upon MafA exclusion indeed causes increasedSomatostatin gene expression, Ad-CMV-Isl-1 was added together with MafAon the 3^(rd) day (treatment C, in Table 1). Indeed, Isl-1 increasedsomatostatin gene expression (FIG. 6E). Ectopic Isl-1 expression(C+Isl-1) caused also increased Somatostatin protein production (FIG.6F) and its co-production in insulin producing cells (FIG. 9, lowerpanel), suggesting that high MafA expression associated by low Isl-1expression is crucial for segregating between insulin and somatostatinproducing cells.

Example 7 Analysis of the Individual Contribution of Pdx-1, Pax4, andMAFA to Liver to Pancreas Transdifferentiation

The sequential characteristics of the transdifferentiation process wereidentified by temporal gain of function studies. Further analysis of theseparate contribution of each of the transcription factors, Pdx-1, Pax4and MafA, to the hierarchical developmental process was performed by arelative and temporal “reduced function” approach. Adult human livercells were treated by the direct temporal and sequential reprogrammingprotocol (treatment C), from which one of the ectopic pTFs was omitted.The omitted pTF was replaced by a control adenovirus carrying β-galexpression at a similar multiplicity of infection. Specifically, adulthuman liver cells were treated by the direct “hierarchical” sequentialinfection order (treatment C, FIG. 3A and Table 1). One singletranscription factor (pTF) was omitted at a time and replaced byidentical moi of Ad-CMV-β-gal. Pdx-1 omission is indicated as (C-Pdx-1),Pax4 omission is indicated as (C-Pax4), and MafA omission is indicatedas (C-MafA).

The functional consequences of separately omitting each of the pTFs'expression were analyzed at the molecular and functional levels (FIGS.6A-6D). Separate Pdx-1 and MafA omission (C-Pdx-1 and C-MafA,respectively) resulted in decreased insulin promoter activation (FIG.6A), ablated glucose response of processed insulin secretion (FIG. 6B)and decreased GLUT2 and GK expression (FIG. 6C). Exclusion of MafAassociated also with decreased expression of the prohormone convertase,PCSK2 (FIG. 6C). On the other hand, exclusion of Pax4 (C-Pax4) did notsignificantly affect insulin promoter activation, nor did it affectglucose-regulated C-peptide secretion. Pax-4 omission was associatedwith decreased GLUT2 and PCSK2 expression (FIG. 6C), possibly suggestingthat the expression of GK is sufficient for obtaining glucose controlability of the hormone secretion.

Analysis of the consequences of the temporal and separate pTF exclusionon the repertoire of the endogenously activated pTF expression wasperformed to explain these developmental alterations. Pdx-1 and Pax4exclusion caused a marked decline in the expression of most other pTFs(including NeuroG3, NKX2.2, NKX6.2, and Pax6), suggesting that theirpotential contribution to increasing transdifferentiation efficiency isrelated to their capacity to activate endogenous pancreatic TFs (FIG.6D). On the other hand, exclusion of MafA did not contribute to furtheractivation of endogenous pTF expression, possibly reflecting its lateand restricted expression only in pancreatic β-cells. On the contrary,MafA contribution to increased insulin promoter activity, prohormoneprocessing and its glucose-regulated secretion was associated only withdecreased Isl-1 expression (FIG. 6D). These data may suggest that MafAis not involved in further promoting the efficiency of endogenous pTFsexpression and liver to pancreas transdifferentiation, but rather inpromoting transdifferentiated cell maturation.

Example 8 Isl-1 Prevents Maturation of Transdifferentiated Cells to βCell Lineage

The effect of MafA on β-cell-like maturation may in part be associatedwith its capacity to repress Isl1 expression. To test this hypothesis,ectopic Isl1 was introduced by adenoviral infection (Ad-Isl1) intransdifferentiated cells. Briefly, adult human liver cells were treatedby the direct “hierarchical” sequential infection order (treatment C)and supplemented by Ad-Isl1 (1 or 100 MOI) at the 3^(rd) day (C+Isl1).

As indicated above, the sequential administration of the three pTFs in adirect hierarchical manner (treatment C) resulted in both increasedtransdifferentiation efficiency and the maturation of the newlygenerated cells along the β-cell lineage. Isl1 was jointly administeredwith MafA on the third day (C+Isl1). Indeed, Isl1 overexpression on thethird day, under the control of a heterologous promoter, resulted insubstantial decrease of insulin gene expression and ablation of glucoseregulated (pro)insulin secretion (FIGS. 7A-7C). The loss ofglucose-sensing ability was associated with diminished GLUT2 expression(FIG. 7C). These results suggest that deregulated Isl1 expression at thefinal stages of the transdifferentiation protocol potentially hampersthe maturation along the β cell lineage, and may account in part for theablated maturation under low MafA expression.

Taken together, these data suggest a crucial obligatory role for directhierarchical expression of pTFs in promoting transdifferentiated livercell maturation along the β cell lineage. Moreover, the sequentialdevelopmental process is associated with both activation and repressionof pTFs that may promote or hamper transdifferentiated cell maturationalong the pancreatic β cell lineage.

Example 9 Pdx-1, Pax4 and MAFA Hierarchical Administration InducesGlucagon and Somatostatin Expression

Transdifferentiation along the endocrine pancreatic lineage results inthe activation of expression of numerous pancreatic hormones. The extentwith which these hormone expression levels are affected by the temporalmanipulation of the pTFs was also investigated. Gene expression ofpancreatic hormones glucagon (GCG) (FIGS. 8A and 8B), somatostatin (SST)(FIGS. 8A, 8D, and 8E) or cells specific transcription factors (FIG. 8C)were determined by quantitative real-time PCR analysis after theindicated treatments.

The transcription of both glucagon (GCG) and somatostatin (SST) geneswas induced by each of the individually expressed pTFs, mainly by Pdx-1and MafA and to a lower extent by Pax4 (FIG. 8A). A further increase inglucagon gene transcription occurred only upon the direct hierarchicaladministration of pTFs (FIG. 4A, see treatment C). Pdx-1 and MafAexerted their effects on glucagon expression in a process associatedwith the activation of the α-cell specific transcription factors ARX andBRAIN4 or ARX alone, respectively (FIG. 8C). Somatostatin geneexpression that remained unaffected by most treatments (FIGS. 8A and8D), was increased when the temporal protocol was concluded by ectopicPax4 expression (E=Pdx-1→MafA→Pax4). This sequential protocol alsoexhibited a deteriorative effect on glucose-regulated (pro)insulinsecretion and was associated by increased Isl1 endogenous expression(FIGS. 3C and 3E). The ablated maturation along the β cell lineage wasassociated with increased somatostatin gene expression and an increasednumber of somatostatin positive cells (FIG. 8F). Many of the cellsexhibited somatostatin and insulin co-localization (data not shown).

Exclusion of each pTF from the hierarchical administration (treatment C)as discussed in Example 6 was also utilized to further investigate therole of the individual pTFs in glucagon and somatostatin expression(FIGS. 8B and 8D). Pax4 exclusion substantially reduced somatostatingene expression, suggesting its potential role in inducing thetranscription of this gene (FIG. 8D). Interestingly, MafA exclusion atthe end of the developmental process also substantially increasedsomatostatin gene expression, suggesting a potential inhibitory effectof MafA on somatostatin gene expression. This effect could be alsoattributed to MafA's capacity to repress Isl1 expression. To addressthis hypothesis, the effect of ectopic Isl1 on somatostatin geneexpression was analyzed. Indeed, Ad-Isl1 administration on the third daytogether with MafA (C+Isl1) increased somatostatin gene expression (FIG.8E), while decreasing insulin gene expression, hormone production andsecretion (FIGS. 8A, 8B and FIG. 7A-7C). Under these experimentalconditions, 40% of the insulin producing cells stained positive forsomatostatin with very few cells expressing somatostatin alone.

These results suggest that part of the maturation of transdifferentiatedcells to β-cells is attributed to MafA expression at the late stages ofthe transdifferentiation process. At this stage, MafA restrictssomatostatin expression in a process associated with its capacity toinhibit Isl1 expression.

FIG. 9 shows the proposed mechanism of pancreatic transcription factorinduced liver to pancreas transdifferentiation. Each of the pTFs iscapable of activating a modest β-cell-like phenotype, in a restrictednumber of human liver cells. The concerted expression of the pTFsmarkedly increases liver to endocrine pancreas transdifferentiation.However the newly generated cells are immature and coexpress bothinsulin and somatostatin. Only sequential administration of the samefactors in a direct hierarchical manner both increasestransdifferentiation efficiency and also the transdifferentiated cellmaturation along the β-cell lineage.

Example 10 Identification of Cell Populations with TransdifferentiationCapacity In Vivo

Cell populations with transdifferentiation capacity were identified invivo in mice. Ectopic expression of the Pdx-1 gene was achieved in micelivers. Despite the uniform expression of the ectopic Pdx-1 gene inabout 40-50% of the cells of the liver (FIG. 10A) (Ferber et al., (2000)Nat Med 6: 568-572, and Ber et al., (2003) ibid) insulin-producing cells(IPCs) in Pdx-1-treated mice in vivo were primarily located close tocentral veins (FIG. 10B), which is characterized by active Wnt signalingand the expression of glutamine synthetase (GS) (FIG. 1C). Theco-localization of GS expression and insulin activation by Pdx-1 alsoindicated that those cells that can activate the GSRE have apredisposition for increased transdifferentiation capacity. Therefore,cell populations predisposed for transdifferentiation can also beidentified by GSRE activation or active Wnt-signaling pathway.

Example 11 Using Adenoviruses to Identify Human Liver Cells Predisposedfor Transdifferentiation

This example demonstrates the use of recombinant adenoviruses toidentify human liver cells that are predisposed fortransdifferentiation. Human liver cells in culture are heterogeneouswith regard to the activation of the intracellular Wnt signaling pathwayand expression of GS. As GS is uniquely expressed in pericentral livercells, therefore the capacity to activate GSRE (GS Regulatory Element)can be used as a selective parameter of isolation of relevant cells.

In addition as the GSRE contains also a STAT3 binding element, thepredisposition of the cells to transdifferentiation could be mediated bythis element. The STAT3 pathway could also be involved in endowing thecells with reprogramming or transdifferentiation predisposition (FIGS.10A-10D, 11, 14A-14E and 19).

Example 12 GSRE Repetitively Targets 13-15% of the Human Liver Cells inCulture

GSRE includes TCF/LEF and STATS binding elements (FIG. 11). Tworecombinant adenoviruses that carry the expression of eGFP gene or Pdx-1genes under the control of GSRE (FIG. 11) operatively linked to aminimal TK promoter have been generated. These adenoviruses drove theexpression of either Pdx-1 (FIG. 12A) or eGFP (FIG. 12B). Both proteinswere repetitively expressed in about 13-15% of the human liver cells inculture suggesting the targeting of a specific population of livercells.

Example 13 GSRE Driven Pdx-1 is More Efficient than CMV Driven Pdx-1 inActivating Insulin Production in Liver Cells

Despite the repetitive expression of GSRE driven PDX-1 only about 13±2%of the cells in culture showed transdifferentiation capacity similar orhigher than that induced by Ad-CMV-Pdx-1, which drives Pdx-1 expressionin 60-80% of the cells in culture (FIGS. 13A-13C). GSRE-activating cellscould account for most of the transdifferentiation capacity of theentire adult human liver cells in culture. Insulin production occurredin 25% of Pdx-1 positive cells upon Ad-GSRE-Pdx-1 treatment compared to1% of the Ad-CMV-Pdx-1 treated cells.

Example 14 Using Lentiviruses to Permanently Label the GSRE+Cells byEGFP

Permanent lineage tracing was performed using Lentivirus constructs. Invitro lineage tracing for GSRE activity was performed by a modified duallentivirus system recently used to trace KRT5 in keratinocytes oralbumin expression in liver cells. This lentivirus system (acollaboration with Prof. P. Ravassard from Université Pierre et MarieCurie Paris, France; FIG. 12A) includes the CMV-loxP-DsRed2-loxP-eGFP(RIG) reporter and an additional lentiviral vector carrying theexpression of Cre recombinase under the control of GSRE and a minimal TKpromoter (generously contributed by Prof. Gaunnz, Germany, FIG. 3A).Thus, GSRE-activating cells are irreversibly marked by eGFP (eGFP+),while the rest of the doubly infected cells are marked by DsRed2(DsRed2+). Ten to fourteen percent of the cells became eGFP+ within lessthan 10 days (FIG. 14B). The cells were separated by a cell sorter(FIGS. 14A-14E) and separately propagated (FIG. 15A). Cultures of eGFP+(GSRE activators) and DsRed2+ cells were generated from 10 differenthuman donors (ages 3-60).

Example 15 eGFP+Cells Consistently Exhibited SuperiorTransdifferentiation Capacity

Human liver cells separated by lineage tracing according to GSREactivity efficiently propagated (FIG. 15A) and were similarlyefficiently infected by recombinant adenoviruses. eGFP+ cellsconsistently exhibited superior transdifferentiation capacity (FIG.16A-16C) manifested by insulin and glucagon gene expression that wascomparable to that of human pancreatic islets in culture (FIG. 16A),glucose regulated insulin secretion (FIG. 16B) and glucose regulatedC-peptide secretion (FIG. 16C). These capacities were consistent and didnot diminished upon extensive cell proliferation, (FIG. 17).

Example 16 Characterization of Cells With Predisposition forTransdifferentiation

To identify the factors that could potentially affect the distincttransdifferentiation efficiencies of the human liver cells, the globalgene expression profile of the two separated populations was comparedusing microarray chip analyses. Human liver cell cultures derived from 3different donors and separated into eGFP+ and DsRed2+ cells werepropagated for 4 passages. The extracted RNA was converted into cDNA andsubjected to microarray chip analysis using the General Human Array(GeneChip Human Genome U133A 2.0 Array, Affymetrix). While most of thegenes were expressed at comparable levels in the separated groups, theexpression of about 800 probes was significantly different (FIG. 18).According to microarray chip analyses, about 100 genes coding formembrane proteins are differentially expressed between thetransdifferentiation-prone (eGFP+) and non-responding (DsRed2+) cells.

Several of these markers are presented in Table 2A and 2B.

TABLE 2A Membrane antigens that are differentially expressed in eGFP+and DsRed2+ cells. High Fold Antigene expression (Log 2) p-valuecommercial antibody ABCB1 DsRed2 −6.363 1.52E−02 BD Biosciences(#557002) ITGA4 DsRed2 −1.979 2.69E−02 R&D system (FAB1354G) ABCB4DsRed2 −4.42 4.62E−02 Abcam (ab24108) PRNP DsRed2 −1.35 4.20E−02eBioscience (12-9230-73) HOMER1 eGFP 1.41 3.25E−04 Biorbyt (orb37754)LAMP3 eGFP 1.285 1.81E−02 BD Biosciences (#558126) BMPR2 eGFP 1.2363.50E−02 R&D system (AF811)

TABLE 2B Cell-surface coding transcripts differentially expressed ineGFP+ vs. DsRed2+ cells Fold change ΔCt Gene EGFP+/DsRed2+ (gene-actin)symbol Gene name cells eGFP+ cells ITGA6 INTEGRIN ALPHA-6 2.82759 8.6DCBLD2 DISCOIDIN, CUB AND 2.4747 12.3 LCCL DOMAIN- CONTAINING PROTEIN 2THBS1 THROMBOSPONDIN-1 2.29441 1.5 VAMP4 VESICLE-ASSOCIATED 1.97484 18.3MEMBRANE PROTEIN 4

FIG. 47 shows the relative expression of the cells surface moleculespresented in Table 2B. Expression levels of specified molecules weretested by Real Time PCR and normalized to beta-actin expression.Microarray data suggested numerous membrane proteins that aredifferential expression between the eGFP+ and the DsRed2+ cells(Fold=eGFP+ differential expression compared to the DsRed2+(log 2). Allthe presented antigens have commercially available antibodies.

Example 17 Wnt Signaling is Active in Cells Predisposed forTransdifferentiation

Liver zonation has been suggested to be controlled by a gradient ofactivated β-catenin levels; while most cells in the liver contain verylow β-catenin activity, the pericentral liver cells express highβ-catenin activity associated with active Wnt signaling. Since Wntsignaling is obligatory for competent β cell activity, the pTFs-inducedpancreatic lineage activation in the liver is restricted to cells that apriori display active Wnt signaling.

GSRE utilized a TCF regulatory element isolated from the 5′ enhancer ofGS. If Pdx-1-induced liver to pancreas transdifferentiation is mediatedin part by the intracellular Wnt signaling pathway, factors thatmodulate the Wnt signaling pathway can also affect transdifferentiationefficiency (FIG. 19).

This data in adult human liver cells suggest that increasingconcentrations of Wnt3a increased Pdx-1-induced glucose-regulatedinsulin secretion, while DKK3 (an inhibitor of the Wnt signalingpathway) completely abolished the effect of Pdx-1 on the process (FIG.19). DKK3 also totally abolished the transdifferentiation capacity ofthe eGFP cells isolated according to their ability to activate GSRE(FIG. 20).

Characterization of Wnt signaling pathway activity in the eGFP+ andDsRed+ cell populations was performed. The APC expression, whichparticipates in β-catenin destabilization, thus diminishing Wntsignaling, was 700% higher in DsRed2+ cells than in the eGFP+ cells(FIG. 21A, in relative agreement with the zonation displayed in vivo).The eGFP+ population has increased activated β-catenin levels (40%)compared to the levels analyzed in DsRed2+ cells (FIGS. 21B and 21C).These data demonstrate that Wnt signaling is active in cells that arecompetent for GSRE activation and have predisposition fortransdifferentiation.

Example 18 Comparing the Efficiency of Transdifferentiation Induced byPax4 and Neurod1

Aim

The aim of this study was to compare the PAX4 and NeuroD1 adenoviruses(Ad-PAX4 and Ad-NeuroD1) in promoting the transdifferentiation processinduced by Ad-PDX-1.

Materials and Methods

The comparison of the transdifferentiation efficiency induced by Ad-PAX4or Ad-NeuroD1 was performed on three naïve cultures (unsorted primaryhepatocyte cells) obtained from human subjects Muhammad, Pedro, andDiego, and four primary hepatocyte cultures following sorting forglutamine synthetase response element (GSRE) activation (GS enriched):Shalosh, Eden, Muhammad and Yam.

Experimental Design

On the first day of the experiment, 300,000 cells were seeded afterviral infection on 100 mm Falcon dish according to Table 3. On the thirdday of the experiment, cells were counted and treated by Ad-MafA andseeded on 3 wells of a 6 wells dish to a final concentration of 100,000cells/well. On the sixth day of the experiment, cells were analyzed forinsulin secretion using a radioimmunoassay. Insulin secretion wasmeasured following incubation of the cells for 15 minutes with either 2mM glucose (low) or 17.5 mM glucose (high) in KRB.

TABLE 3 Summary of the different combination of adenoviruses used forcomparing the role of PAX4 and NeuroD1 in the transdifferentiationprocess induced by PDX1. Day1 Day3 1 Ad-Null 1300moi 2 Ad-PDX1 500moi +Ad-NeuroD1 250moi Ad-MafA 50moi 3 Ad-PDX1 500moi + Ad-PAX4 250moiAd-MafA 50moi

Results

The results are summarized in Tables 4 and 5 and FIGS. 24A-24B and25A-25D.

TABLE 4 Summary of the final calculations of total insulin (INS)secretion per hour (ng INS/hr) comparing the role of PAX4 and NeuroD1 inthe transdifferentiation process induced by PDX1. Total ng/h Averageng/h SE Samples Low High Low High SE 2 mM 17.5 mM Control Shalosh GS 00.380923 0.166896 0.524835 0.101164 0.112632 enriched Eden Green0.197364 0.758004 Muhammad 0 0.721483 GS enriched Yam GS 0 0.397280.034227 0.430182 0.032654 0.114352 enriched Max GS 0 0 enriched Eden GS0.008 0.3234 enriched Muhammad 0.245521 1.069337 0.432233 0.7141420.254312 0.244889 Naive Pedro Naïve 0.935318 0.244501 Diego Naive0.115859 0.828589 PDX1 + NeuroD1 Shalosh GS 0.102627 1.138869 0.143971.601043 0.057159 0.351225 enriched Eden Green 0 1.500592 Muhammad0.027635 1.048397 GS enriched Yam GS 0.217733 4.162756 0.119999 1.9006020.060742 0.480813 enriched Max GS 0 2.177 enriched Eden GS 0.372 1.376enriched Muhammad 0 1.411349 0.191913 1.001924 0.137961 0.23494 NaivePedro Naïve 0.459557 0.996881 Diego Naive 0.116183 0.59754 PDX1 + PAX4Shalosh GS 0.381611 0.491117 0.351915 1.301016 0.087502 0.275093enriched Yam GS 0.056133 0.785065 enriched Muhammad 0.302323 2.249145 GSenriched Max GS 0.057 2.744 0.223414 1.455865 0.137343 0.393614 enrichedEden GS 0.32 1.01 enriched Muhammad 0.89452 1.376825 0.566084 1.0429330.037142 0.370843 Naive Pedro Naïve 0.447356 0.4218 Diego Naive 0.3563761.330176

TABLE 5 Summary of the final calculations of total insulin (INS)secretion per million cells per hour (ng INS/10⁶ cells/hr) comparing therole of PAX4 and NeuroD1 in the transdifferentiation process induced byPDX1. Average ng/h/10{circumflex over ( )}6 cells ng/h/10{circumflexover ( )}6 cells SE Samples 2 mM 17.5 mM SE 2 mM 17.5 mM Control ShaloshGS 0 5.355152 0.671145 4.501346 0.271034 0.877392 enriched Eden Greeen1.265156 4.859 Muhammad 0 5.900258 GS enriched Yam GS 0 1.91 0.2341933.835735 0.207456 0.955189 enriched Max GS 0 0 enriched Eden GS 0.144.99 enriched Muhammad 2.00425 8.190631 1.54505 5.832567 0.3058421.829411 Naive Pedro Naïve 1.665405 2.230751 Diego Naive 0.9654947.07632 PDX1 + NeuroD1 Shalosh GS 1.345204 13.19027 2.016969 16.199331.042752 2.502689 enriched Muhammad 0.310173 14.16339 GS enriched EdenGreen 0 12.82557 Yam GS 1.136 21.7188 1.926396 18.43701 1.3879313.003895 enriched Max GS 0 16.864 enriched Eden GS 8.767 31.86 enrichedMuhammad 0 17.52215 2.198114 11.72398 1.841633 3.875939 Naive PedroNaïve 5.856674 13.28074 Diego Naive 0.737667 4.369039 PDX1 + PAX4Shalosh GS 5.984453 7.723947 3.954761 14.31825 0.917087 2.523347enriched Yam GS 0.421 5.888 enriched Muhammad 2.468333 18.063 GSenriched Max GS 0.52 25.753 3.050957 14.85359 1.22808 3.634948 enrichedEden GS 5.861 16.84 enriched Muhammad 8.187757 14.31595 5.46110113.42601 1.410137 4.709221 Naive Pedro Naïve 4.721848 4.860915 DiegoNaive 3.473699 21.10115

A detailed comparison was made between the two pancreatic transcriptionfactors. The comparison was made on mixed populations of naïve primaryhepatocytes and hepatocyte populations enriched by sorting for enhancedGS expression (GS enriched).

FIGS. 24A-24B and 25A-25D present the tabulated data as bar graphs.

Insulin secretion measurements revealed that there is no statisticaldifference in the transdifferentiation induced using PAX4 or NeuroD1.This conclusion was true for both naïve cells and enriched GS cells. Itwas not only the averages of the enriched GS populations and naïve cellsthat showed the same trends, when examining the results of the sameculture Muhammad naïve and Muhammad GS enriched, the same results wereobtained (demonstrating the ability of the GS enriched population toserve as a model system for the transdifferentiation process).

Previous results showed that GS enriched populations had a clearadvantage over the full hepatocyte primary culture with regard totransdifferentiation efficiency. It was therefore, surprising that theGS enriched population and the unsorted population of Muhammad showedsimilar results (no statistical significance). However, it should bementioned that there was a difference in the passage number of bothpopulations. The GS enriched population was examined in passage 19 andthe naïve population was examined in passage 7. These results should notbe addressed as a failure of the GS enriched population to undergoeffective transdifferentiation but as the GS enriched population'sability to undergo transdifferentiation in high passages that the naïvecells may not be able to achieve.

There were no significant differences in the cell death of cellsincubated with PAX4 compared to cells incubated with NeuroD1. The onlydifference that was evident was of control group (untreated/Ad-Null)compared to the treated groups (Ad-PAX4/Ad-NeuroD1). This is seen by thesame conclusions reached for PAX4 and NeuroD1 whether examining theresults for Total Insulin or for ng INS/10̂6 cells/hr.

The one difference observed was when calculating thetransdifferentiation efficiency (percent of positivetransdifferentiation obtained when using the specific adenovirus). ForAd-NeuroD1 the efficiency was 87.5% (7 positive transdifferentiation outof 8 experiments) and for Ad-PAX4 it was 71% (5 positivetransdifferentiation out of 7 experiments).

Conclusion

Both Ad-PAX4 and Ad-NeuroD1 support similar transdifferentiation ofhepatocytes.

Example 19 Determining the Optimal Protocol for the TransdifferentiationProcess

Aim

The aim of this study was to compare the transdifferentiation efficiencyof the full hierarchy (1+1+1 protocol), with the 2+1 protocol, and withsimultaneous infection with all three adenoviruses.

The Test System

The different transdifferentiation protocols were examined on threeprimary cultures of human liver cells, Leon, Muhammad, and Pedro grownin DMEM 1 g/L glucose. After viral infection cells were grown in DMEM 1g/L glucose media supplemented with 5 nM Exendin-4, 20 ng/ml EGF and 10mM Nicotinamide.

Experimental Design

The different transdifferentiation (TD) protocols were examinedaccording to the Table 6 below. Briefly, on the first day of theexperiment 300,000 cells were seeded after viral infection on 100 mmFalcon dish according to Table 6 below for protocols A (Null), B (2+1)and E (Hierarchy 1+1+1). On the second day of the experiment 100,000cells were seeded on 6 wells dish for protocol C (3 factorssimultaneously) and 70,000 cells were seeded on 6 wells dish forprotocol D (3 factors simultaneously). On the third day of theexperiment, cells were counted and treated by Ad-MafA (protocols B andE) and seeded on 3 wells of a 6 wells dish to a final concentration of100,000 cells/well.

TABLE 6 Day 1 Day 2 Day 3 Day 6 A Null (1300moi) GSIS* B PDX1 1000moi +NeuroD1 MafA GSIS 250moi 50moi C PDX1 1000moi + GSIS NeuroD1 250moi +MafA 50moi D PDX1 1000moi + GSIS NeuroD1 250moi + MafA 50moi E PDX1 (E4)1000moi NeuroD1 250moi MafA GSIS 50moi *GSIS—Glucose stimulated insulinsecretion

On the sixth day of the experiment, cells underwent secretion analysisin the presence of 2 mM glucose (low) or 17.5 mM glucose (high) (FIGS.26A-26C). Insulin secretion was measured following incubation of cellsfor 15 minutes with 2 mM glucose or 17.5 mM glucose in KRB.

Results and Analysis

The present study sought to determine the optimal protocol for thetransdifferentiation process. In the traditional hierarchy protocol(1+1+1), cells are treated sequentially with three transcriptionfactors: PDX1 on day 1, NeuroD1 on day 2 and MafA on day 3. In an effortto develop an efficient and easier protocol, the transdifferentiationefficiency of the traditional protocol, was compared with the 2+1protocol and simultaneous treatment with all three transcription factorspresent.

The read out assay for this examination was insulin secretion. Accordingto knowledge in the field, all treatments should have presented similarlevels of insulin secretion, as differences in efficiency should bepresented only in the maturation of the cells, for example as measuredby C-peptide secretion. However, in the present experiments there wereunexpected differences in transdifferentiation efficiency as clearlyseen by the insulin secretion measurements (FIGS. 26A-26C). The bestresults were obtained in the 2+1 protocol. These results werestatistically significant, as shown in Table 5 below.

TABLE 7 p-value (t-Test) for the comparison of the differenttransdifferentiation protocols presented in Table 4 above. 3 factors 3factors Hierarchy (70K) (100K) 2 + 1 2 + 1 0.06691407 0.045611240.017915142 3 factors 0.223713506 0.35910095 0.017915142 (100K) 3factors (70K) 0.376772188 0.35910095 0.04561124 Hierarchy 0.3767721880.223713506 0.06691407

The p-value of the 2+1 protocol and the hierarchy protocol issignificant but relatively high. The simultaneous treatment with allthree factors presented the lowest results even though two seedingdensities were examined (not significant in comparison to the hierarchyprotocol).

Example 20 Industrialization of Liver Cell Proliferation Process fromPetri Dish to the Xpansion Multiplate Bioreactor

Aim

A bioprocess in cells dishes for preclinical applications was developedthat included 2 main steps: liver cell proliferation followed by livercell transdifferentiation into insulin producing cells. For treatment ofpatients in human clinical trials, it is anticipated that a doserequirement of about 1 billion cells per patient would be used toameliorate hyperglycemia in Type 1 diabetes. Such a production scalewould require large culture surface area, which the Cell Culture DishProcess (FIG. 27 top) manufacturing strategy does not provide. Thus thegoal of this study was to industrialize the cell based Cell Culture DishProcess using the XPANSION platform (bioreactor system; PallCorporation, USA).

Materials and Methods

The materials used are listed below:

-   -   i. Biological materials: Human adult liver-derived cells        (primary culture).    -   ii. Growth medium: Dulbecco's Modified Eagle Medium (DMEM; Life        Technologies Cat. 21885-025) supplemented with 10%        heat-inactivated fetal bovine serum (FBS; Life Technologies Cat.        10500-064), 1% Penicillin-Streptomycin-Amphotericin B (100×)        (Lonza Cat. 17-745E) and 5 nM Exendin-4 (Sigma-Aldrich Cat.        E7144)    -   iii. Other reagents: Dulbecco's Phosphate Buffered Sales (DPBS;        Lonza Cat. 17-512Q) and TrypLE♦ Select (Life Technologies Cat.        12563-029).    -   iv. Cell culture support: CellBIND♦ CellStack♦ 2-, 5- &        10-chamber (Corning Cat. COS-3310, COS-3311 & COS-3320),        Xpansion 50 plates (XP-50) bioreactor (Cat. XPAN050000000) and        Xpansion 200 plates (XP-200) bioreactor (Cat. 810155).    -   v. Cell Recovery Kit for Pall's continuous centrifuge (item        6100043)    -   vi. Centrifuge control: Cell Recovery System control in 500 mL        centrifuge bowl

The methods follow the Process Flow Chart presented in FIG. 27. Briefly,pre-cultures were performed as traditional multi-tray cultures. Cellswere used in the Xpansion bioreactor(s) at passage 14 & 15. Thebioreactor system used is a closed system for reduced risk ofcontamination. Multi-tray cultures were performed in parallel to theXpansion culture as a cell growth control with controller set points ofpH: 7.3-7.6 and dissolved oxygen (DO): maintained above 50%. The targetseeding density was 4,000 cells/cm² at each passage.

Culture duration was 7-9 days with a medium exchange applied every 2-4days (XP-50: Days 4, 6 and 8—XP-200: Days 4 and 7) to maintain glucoselevel above 0.5 g/L throughout the culture.

Results

The results presented here show the successful scale-up of the humanliver-derived cell amplification phase from Petri dishes to the Xpansion200 bioreactor (Pall Corporation, USA).

Cell Growth—

The cell expansion profile presented in FIG. 28 clearly demonstratesthat cells are in exponential phase of growth from the firstpre-cultures steps to the final bioreactor (XP-200) culture. Within 4passages, cells were amplified from 2 million to ˜1.8 Billion,representing a 1,000-fold biomass increase. Therefore, feasibility oflarge-scale production of human liver-derived primary cells has beenclearly demonstrated, and a target of 1 billion cells/patient, and evennearly 1.8 billion cells/patient per XP-200 was achieved.

Passage 1 was carried out in CellStack10 (CS10), passage 2 was carriedout in 2×CS10, passage 3 was carried out in an XP50, and passage 4 wascarried out in an XP200.

Population doubling time (PDT) comparison revealed that the humanliver-derived primary cells proliferated faster in the Xpansionbioreactor than in the traditional multi-tray system (FIG. 29).Harvested cell densities were around 15,000 cells/cm² in the Xpansion 50bioreactor, and 14,000 cells/cm² in the Xpansion 200 bioreactor,representing ˜160% of their respective multitray controls. Bettercontrol of the culture environment (pH, DO) is the main hypothesis toexplain this result.

pH, Dissolved Oxygen, and Temperature Control Trends in the XpansionBioreactor—

pH and DO were maintained in their respective expected ranges (FIG. 30).DO was maintained up to 50% of air saturation throughout the wholeprocess, and pH decreased progressively from 7.4 to 7.2 during the last2 days of each culture due to high cell number at the end of theprocess. Similar trends were observed during both cultures demonstratinga good reproducibility and scalability.

Microscopic Observation Using the Ovizio Holographic Microscope (OvizioImaging Systems, Brussels/Belgium)

Cell confluence and morphology are key parameters to monitor in celltherapy processes. To this end, a microscope that allows observation ofthe top ten plates in the Xpansion bioreactor was used.

Micrograph images presented in FIGS. 31A-31D confirm the homogeneousdistribution of human liver-derived primary cells throughout theXpansion plates. Cell confluence was determined to be approximately 90%after 9 days of culture, and estimated to be equivalent in both theXP-50 and XP-200 bioreactors (FIGS. 31A and 31B). At both the 50- and200-plate scale, confluence in the Xpansion system was slightly higherthan that from the control multi-tray system. These images alsodemonstrate that the cell morphology was not affected by successiveculture in the Xpansion system, or by continuous centrifugation used forcell recovery. Control cells grown using a multi-tray process are shownin FIGS. 31C and 31D. Data demonstrated that human liver-derived primarycell proliferation using the Xpansion bioreactor did not alter thetransdifferentiation properties or the insulin secretion profile oftransdifferentiated cells (Data not shown).

Conclusion

Bioreactors were successfully used to scale-up the human adultliver-derived cells proliferation process. The results herein show thatby using a process including a bioreactor platform, cells could bereliably amplified from 1 million up to 1.8 billion cells. This level ofscale up potentially makes available 1.8 billion cells foradministration to patients during a cell-based autologous therapytargeting diabetes. This compares to the only 7 million cells producedusing a cells dish process, e.g., petri dishes (data not shown)Importantly, the process using bioreactors preserved cell viability,potential for transdifferentiation, and the cell's insulin secretionprofile.

Example 21 Protocol for Producing Autologous Insulin Producing (AIP)Cells for the Treatment of Diabetes

Aim

The aim of this study was developing an industrial scale protocol forproducing autologous insulin producing (AIP) cells from non-β pancreaticcells for the treatment of diabetes. By correcting functionally formalfunctioning pancreatic insulin producing β-cells with new functionaltissues generated from the patient's own existing organs, a cell-basedautologous therapy could successfully target diabetes in a subject.

The protocol presented herein employs a molecular and cellular approachdirected at converting human liver derived cells into functionalinsulin-producing cells by transcription factors inducedtransdifferentiation (FIG. 32). This therapeutic approach generatesAutologous Insulin Producing (AIP) cells on an industrial scale,overcoming the shortage in tissue availability from donors.

Overview of the Protocol

FIG. 33 provides an overview of the protocol provided here,demonstrating an approximate time from biopsy to finished product of6-weeks, along with approximate cell numbers at each step. FIG. 34presents a flowchart of the human insulin producing cell product cellproduct manufacturing process, which may in one embodiment be autologousor allogeneic insulin producing cells (AIP). Details are provided below.

Obtaining Liver Tissue Step 1 of FIG. 34

Liver tissue was obtained from adult human subjects. All liver tissueobtained were received under approval of the Helsinki Committee of theMedical Facility. Accordingly, all liver tissue donors signed aninformed consent and Donor Screening and Donor Testing was performed toensure that biopsies from donors with clinical or physical evidence ofor risk factors for infectious or malignant diseases were excluded frommanufacturing of human insulin producing cells.

Liver biopsies were obtained in an operating theatre by qualified andtrained surgeons. A biopsy of the size of about 2-4 g of liver tissuewas taken from eligible patients and transported at 2-8° C. inUniversity of Wisconsin (UW) solution in a sterile bag to the GMPfacility.

In Vitro Culture/Steps 2 and 3 of FIG. 34

At the manufacturing site, liver biopsies were processed as for adherentcells. Briefly, biopsy tissue was cut into thin slices and digested bycollagenase type I for 20 min at 37° C. Subsequently, cells wererepeatedly digested with trypsin in order to obtain isolated singlecells; initial experiments had shown that approx. 0.5×10⁶ cells can beisolated per gram biopsy.

Cells were then expanded ex vivo in cells medium supplemented with 10%FCS, Exendin-4 and a mix of antibiotics (Penicillin, Streptomycin andAmphotericin B). Cells were passaged at 37° C. in a humidifiedatmosphere of 5% CO₂/95% air (up to 20 passages) using pre-treatedFibronectin-coated tissue culture dishes. Medium was changed dailyduring the first three days post biopsy plating to remove non-adherentcells followed by twice a week, after the first cell passage. At thetime of the first cell passage at least one aliquot of cells wascryopreserved (see below; Optional Step of FIG. 34).

Cells were passaged 1:3 using trypsin until the desired number of cellswas generated (about 1-3 billion cells, within about 4 to 7 weeks).Expansion of cells included use of Multi-plate systems as described inExample 20 and shown in FIG. 33 at approximately week 4 through weeks 7.(Step 3 of FIG. 34)

Human liver cells that adhered to the tissue culture plates underwentepithelial to mesenchymal transition (EMT) and efficiently proliferated.Close to 100% of these EMT-like cells displayed the known mesenchymalcharacteristics (CD29, CD105, CD90 and CD73) but also expressed adulthepatic markers such as albumin and AAT. The cells neither expresshepatoblast nor “stemness” markers. Table 8 below shows the results ofanalysis of these EMT-like cultured liver cells for the presence ofmesenchymal, hematopoietic, and hepatic markers on the cultured livercells prior to transdifferentiation (TD).

TABLE 8 Before Transdifferentiation Specification Mesenchymal markersCD105, CD73, CD90, CD44 >95% Haemapoeitic markers <2% Hepatic markersAlbumin >80% AAT >60%

The percentages shown in Table 8 are at low passage number.

Cryopreservation of Passage 1 Cells (FIG. 34)

Briefly, Passage 1 cells were cryopreserved in DMEM supplemented with10% FBS and 10% DMSO in 2 ml cryovials (minimum of 0.5×10⁶ cells). It isrecommended to cryopreserve cells at the earliest passage possible.Frozen cells were first stored at −70° C. for 24-48 hours and thentransferred to liquid N₂ for long term storage.

Thawing of Cryopreserved Cells (FIG. 34)

Cryopreserved cells were thawed using well-known methods in the art.Briefly, vials were removed from liquid N₂ and allowed to slowly thawuntil sides were thawed but center was still frozen. Cells were gentlytransferred to tissue culture plates. Once cells have attached to theplate, in vitro processing (Steps 2 and 3 of FIG. 34) to expand the cellculture was performed.

Select Predisposed Liver Cells (FIG. 34)

An option at Step 3 of FIG. 34 is to sort the Primary Liver Cells inorder to enrich for cells predisposed to transdifferentiation. Forexample, cells could be sorted for glutamine synthetase response element(GSRE) activation (GS enriched cells), as described herein above inExamples 10-15. Alternatively, cells could be enriched for having anactive Wnt signaling pathway, wherein they are predisposed to respond toWnt signaling, as described herein above in Example 17. In addition,cells could be enriched by monitoring increases or decreases ofexpression of certain genes, for example decrease in expression ofABCB1, 1TGA4, ABCB4, or PRNP, or any combination thereof, or increasesin expression of HOMER1, LAMP3, BMPR2, ITGA6, DCBLD2, THBS1, or VAMP4,or any combination thereof, as described herein above in Example 16. Thecell population could be treated with lithium, as described in Example23, in order to enhance the predisposition of cells totransdifferentiation. Following enrichment for predisposition totransdifferentiation, cells are used at Step 4 of FIG. 34.

Trans-Differentiation (Step 4 of FIG. 34)

For trans-differentiation cells were grown in trans-differentiationmedium for an additional 5 days. Trans-differentiation medium isDulbecco's minimal essential medium (1 g/l of glucose) supplemented with10% FCS, Exendin-4, Nicotinamide, EGF and a mix of antibiotics(Penicillin, Streptomycin and Amphotericin B).

Two different protocols were used for transdifferentiation of cells.Cells were transdifferentiated using the Hierarchy (1+1+1) sequentialprotocol or using the 2+1 protocol. Examples of each method are providedbelow.

Hierarchy (1+1+1) Sequential Protocol

Ex vivo expanded liver cells were then sequentially infected with 3serotype-5 recombinant replication-deficient adenovirus vectors, eachcarrying the human gene for one of the pancreatic Transcription Factors(pTFs), PDX-1, Neuro-D or MafA, under the control of the cytomegalovirus(CMV) promoter. The 3 human pTF genes had been inserted into the samebackbone of FGAD vectors under the control of the CMV promoter. The CMVpromoter is a heterologous promoter that is usually turned off within3-4 weeks after infection. Nevertheless the short-term expression of theectopic pTF genes was sufficient to induce the endogenous humanhomologs.

FGAD vectors were selected as an optimal gene delivery tool for inducingdevelopmental redirection. Examples above demonstrated that introductionof these ectopic genes into primary adult human liver cells acts asshort term triggers for an irreversible process of reprogramming ofadult cells. On the other hand, the recombinant adenoviruses wererelatively safe as they do not integrate into the host genome andtherefore do not disrupt the host sequence of genetic information. PDX-1induces epigenetic alterations in the chromatin structure, thus allowingthe activation of otherwise silent genetic information, while turningoff the host repertoire of expressed genes (compare the results ofTables 8 and 9).

The transdifferentiation process was performed using a closed automaticXpansion bioreactor system (Pall Life Sciences), following the flow ofsteps presented in FIG. 33. The bioreactor system was used forcultivation of cell cultures, under conditions suitable for high cellconcentrations. The bioreactor system was constructed of two mainsystems, a control system and a bioreactor itself (vessel andaccessories).

The parameters of the process were monitored and controlled by thecontrol console which included connectors for probes, motor and pumps,control loops for Dissolved Oxygen (DO), pH, a gases control system andplace in the incubator for temperature control. The controlled processparameters (such as temperature, pH, DO etc.) could be displayed on theoperator interface and monitored by a designated controller.

Cell Culture Growth Procedure in the Bioreactors

250±50×10⁶ cells were seeded in a sterile XP-200 bioreactor. The growthmedium in the bioreactor was kept at the following conditions: 37° C.,70% Dissolved Oxygen (DO) and pH 7.3. Filtered gases (Air, CO₂, N₂ andO₂) were supplied as determined by the control system in order to keepthe DO value at 70% and the pH value at 7.3. Growth media was changedwhen the medium glucose concentration decreased below 500 mg/liter. Themedium was pumped from the feeding container to the bioreactor usingsterile silicone tubing. All tubing connections were performed with atube welder providing sterile connectors. A sample of the growth mediumwas taken every 1-2 days for glucose, lactate, glutamine, glutamate andammonium concentration determination. The glucose consumption rate andthe lactate formation rate of the cell culture enabled to measure cellgrowth rate. These parameters were used to determine the harvest timebased on accumulated experimental data.

Harvest of the Cells from the Bioreactor

The cell harvest process started at the end of the growth phase (8-16days). The culture was harvested in the Class-100 laminar area asfollows:

The bioreactor vessel was emptied using gravitation via tubing to awaste container. The bioreactor vessel was then refilled with 22 Lpre-warmed PBS (37° C.). The PBS was drained via tubing by pressure orgravity to the waste bottle. The washing procedure was repeated twice.

In order to release the cells from the surface, 22 L pre-warmed to 37°C. of Trypsin-EDTA (Trypsin 0.25%, EDTA 1 mM) was added to thebioreactor vessel. 500 ml FBS was added to the bioreactor vessel and thecell suspension was collected to a sterile container. Cell suspensionwas centrifuged (600 RPM, 10 min, 4° C.) and re-suspended in culturemedia.

Hierarchy (1+1+1) Viral Infection Steps

The ectopic transgenes were sequentially administered by recombinantadenoviruses on three successive days. Sequential administration of theectopic genes has been documented to both increase thetrans-differentiation efficiency and to increase the maturation of thecells, specifically along the β cell lineage and function.

The trans-differentiation procedure took approx. 7 days, at the end ofwhich cells are washed to remove the un-incorporated recombinantadenoviruses. Briefly:

On day 1, resuspended cells were infected with the PDX-1 adenoviralvector using an MOI of 1,000. Cells were then seeded onto culture dishesare incubated overnight in a humidified 37° C. incubator supplied with5% CO₂.

On day 2, cells were detached from culture dishes using trypsin andresuspended. Resuspended cells were infected with the NeuroD1 adenoviralvector using an MOI of 250. Cells were then seeded onto culture dishesare incubated overnight in a humidified 37° C. incubator supplied with5% CO₂.

On day 3, cells were detached from culture dishes using trypsin andresuspended. Resuspended cells were infected with the MafA adenoviralvector using an MOI of 50. Cells were then seeded onto culture dishesare incubated for three days in a humidified 37° C. incubator suppliedwith 5% CO₂.

Cells were then recovered and analyzed for markers and glucose regulatedprocessed insulin secretion. Control cells included those propagated andincubated following the same protocol but without addition ofadenovirus.

Materials and Experimental Methods

FACS analysis of membrane markers—cells were stained with monoclonalantibodies as follows: 400,000-600,000 cells were suspended in 0.1 mlflow cytometer buffer in a 5 ml test tube and incubated for 15 minutesat room temperature (RT), in the dark, with each of the followingmonoclonal antibodies (MAbs):

Ab Andibody full name Company Cat. No. PDX1 BD Pharmingen ™ PE Mouseanti-PDX-1 BD 562161 Human/Mouse PDX-1/IPF1 Phycoerythrin MAb R&DSystems IC2419P Human/Mouse PDX-1/IPF1 Allophycocyanin Mab R&D SystemsIC2419A NEUROD1 BD Pharmingen ™ PE Mouse Anti-NeuroD1 BD 563001 BDPharmingen ™ Alexa Fluor ® 647 Mouse anti-NeuroD1 BD 563566 MAFAAnti-KLRG1 (MAFA-)-PE-Vio770, human (clone; REA261) Miltenyi Biotec130-103-641 Anti-KLRG1 (MAFA)-APC-Vio770, human (clone: REA261) MiltenyiBiotec 130-103-642 Vimentin BD Pharmingen ™ PE Mouse Anti-Human VimentinBD 562337 BD Pharmingen ™ Alexa Fluor ® 488 Mouse Anti-Human Vimentin BD562338 E-Cadherin BD Horizon ™ BV421 Mouse Anti-E-Cadherin BD 564186 BDPharmingen ™ PE Mouse anti-E-Cadherin BD 562526 BD Pharmingen ™ AlexaFluor ® 488 Mouse Anti-Human CD324 (E-Cadherin) BD 563570 BDPharmingen ™ PerCP-Cy ™5.5 Mouse Anti-Human CD324 (E-Cadherin) BD 563573BD Pharmingen ™ Alexa Fluor ® 647 Mouse Anti-Human CD324 (E-Cadherin) BD563571 BD Pharmingen ™ PE Mouse Anti-Human CD324 (E-Cadherin) BD 562870BD Horizon ™ PE-CF594 Mouse Anti-Human CD324 (E-Cadherin) BD 563572

Harvesting AIP cells (Step 5 of FIG. 34) Cells were then washed twicewith flow cytometry buffer, resuspended and analyzed by flow cytometryusing an FC-500 flow Cytometer (Beckman Coulter). Negative controls wereprepared with relevant isotype fluorescence molecules.

Packaging and Release

At the end of manufacturing, AIP cells will be packed for shipment andreleased at the manufacturing site. It is planned to ship AIP cells at2-8° C. to the hospitals.

Results of Hierarchy (1+1+1) Protocol

The adenoviral infection of the cells resulted in transient expressionof the transgenes, which triggers permanent induction of endogenousgenes, resulting in stable transdifferentiation to AIP cells (data notshown). As a result, there was no modification or insertions of viralDNA in the final product.

Analysis of Harvested AIP Cells (Step 6 of FIG. 34)

An analysis of the transdifferentiated liver cells (AIP cells) for thepresence of mesenchymal, hematopoietic, and hepatic markers is presentedin Table 7. Negative markers include hematopoietic markers.

TABLE 9 % Negative % CD105 % CD73 % CD90 % CD44 markers 99.32 99.8599.55 99.77 0.93 98.75 99.71 99.67 99.70 0.73 97.89 98.71 99.80 99.770.94 96.77 98.60 99.50 99.64 0.58

While variability was noted across different patient samples in Xpansionbioreactors, in all cased cell density of harvested cells was greatlyincrease as compared with the starting culture (FIG. 35).

The harvested AIP cell product was analyze to identify expression ofnumerous markers. Identity was by RT-PCR and FACS. The results presentedin Tables 10 and 11 below show the fold increase of endogenousexpression of β-cell pancreatic marker genes including PDX-1, NeuroD,MafA, Pax4, Nkx6.1 and insulin.

TABLE 10 Fold increase RT-PCR (over control) Pdx1 >10⁵ NeuroD >10⁴ MafA>10³ Insulin >10¹

TABLE 11 Fold increase RT-PCR (over control) Glucagon >10² Somatostatin>10¹ Nkx6.1 >10¹ Pax4 >10¹

The bar graphs presented in FIGS. 36A and 36B show the typical resultsobtained following use of the hierarchy protocol. A comparison oftransdifferentiated liver cells (AIP cells) with pancreatic cells andthe control population of non-transdifferentiated liver cells ispresented wherein it can be seen the AIP cells show a significantincrease in pancreatic cell markers compared with control.

The result of further characterization of the cells for hepatic versuspancreatic phenotype of function of the AIP cells is presented in Table12 below. The significant decrease of hepatic markers in PDX-1 cellscombined with the increase of pancreatic cell markers indicatessuccessful transformation of liver cells to cells having phenotype andfunction of pancreatic β-cells.

TABLE 12 AIP cells product specification, as identified by FACS AfterTrans-differentiation Specifications Hepatic markers in Pdx- <1% 1 +positive cells Each ectopic pTF >80% Insulin/c-peptide >10% NKX 6.1 >10%Glucagon >10%

Analysis for dead cells within the population of harvested AIP cellsshowed that less than 20% of the cells were dead (data not shown).

The harvested AIP cell product was also analyze for function secretionof insulin. FIG. 37 shows AIP cell product Potency (glucose regulatedsecretion of insulin as measured using ELISA). The AIP cell producttested represents a transdifferentiated population of cells that hadbeen expanded in an XP-200 bioreactor. Insulin secretion was measured byGSIS (glucose stimulated insulin secretion at low (2 mM) and high (17.5mM) glucose concentrations with KRB+0.1% BSA RIA-grade, or recombinantBSA). Results are presented as ng insulin per million cells per hour andshow the significant increase of response of AIP cells.

2+1 Transdifferentiation (TD) Protocol

FIG. 38 presents “2+1” TD protocols using Xpansion bioreactor systems aswell as a process control. The results of using the “2+1” TD protocol incombination with a multi-system bioreactor demonstrated the feasibilityof this protocol, which efficiently produced AIP product cells. Thefirst infection was performed at day 3 using either an adenoviral vectorcomprising a nucleic acid that encoded PDX-1 and NeuroD1 polypeptides ontwo adenoviral vectors—one comprising a nucleic acid encoding PDX-1 andthe other comprising a nucleic acid encoding NeuroD1. The MOI for PDX-1as 1:1,000 and for NeuroD1 was 1:250. Cells were then incubated for 3days and a second infection was performed on day 6 using an adenoviralvector comprising a nucleic acid encoding MafA (1:50 MOI). The cellswere harvested two days later at day 8 and screened for quality controlmarkers, similar to that described above when the hierarchy (1+1+1)protocol was used.

Observation of cell cultures at the time of the second infection (day 6)showed similar confluences independent of the conditions used (FIGS.39A-39D and 40A-40B). At the time of final harvest cells processed underCTL (control) conditions presented slightly higher cell confluence thanother conditions (FIGS. 41A-41D). Differences in cell densities were duemainly to different seeding densities, and cell recovery yields andmortality on days following infection.

The insulin content of harvested cells was assayed and the resultspresented in FIG. 42 demonstrates increased insulin content (microInternational Units/million cells) for cells transdifferentiated underall three 2+1 protocols tested, as compared with controls that wereuntreated (not infected with viral vectors comprising nucleic acidsencoding PDX-1, NeuroD1, and MafA). The process CTL condition presentedexpected trend yielding significantly higher insulin content thanuntreated cells (˜2.5× higher). The Xpansion CTL condition alsopresented expected trend wherein treated cells presented significantlyhigher insulin content than untreated cells (˜1.7× higher). Cellstransdifferentiated in the Xpansion 10 system presented similar insulincontent than treated cells of the Xpansion CTL condition (˜1.7× higherthan untreated control)

Use of the “2+1” transdifferentiation protocol was efficient (reducedstep number and opportunities for cell lose) in producing AIP cellproduct with significantly higher insulin content than untreated livercells.

Purity Assays

Purity assays were developed to ensure that more than 90% of the cellsduring the expansion and transdifferentiation steps have a mesenchymalstem cell (MSC)-like phenotype (See above in Methods). These purityassays were used independent of the protocol used fortransdifferentiation. Cultivated MSCs should stain positive for CD73,CD90, CD105, and CD44. In addition, MSCs should be negative for CD45,CD34, CD14 or CD11b, CD19 or CD79#, and HLA-DR surface molecules.Previous results (FIGS. 44A and 44B) demonstrated that MSC markers werestable over time and during transdifferentiation of liver cells. Resultsshowing the MSC-like phenotype of AIP cells are presented in Tables 6and 7. Both flow cytometry and immunofluorescence assays were used toexamine these parameters.

Example 22 Analysis of Digestion Methods

Objective

The objective of this study was to verify that different digestionmethods do not impact the ability of liver cells to be transduced byadenoviruses.

Methods

Briefly, liver cells were infected with Ad.CMV.GFP and the expression ofGFP was measured after 96 hours. Liver cells were transduced with 10,100, and 500 moi of Ad5.CMV.GFP virus or left untreated. After 96 hours,GFP expression was measured by fluorescent microscopy (FIG. 45A, FIG.46A) and by FACS (FIGS. 45B-45C, FIGS. 46B-46C).

Results

FIGS. 45A-45C shows the efficiency of transduction of BP001 cells,derived from digestion of livers with Serva and Worthingtoncollagenases. Although the percentage of transduced cells was similar,liver digested with Serva collagenase produced more GFP than liverdigested with Worthington collagenase, as shown by the GFP fluorescentintensity (FIGS. 45B and 45C). Similarly, transduction efficiency ofTS001 cells was not impacted by the use of Serva collagenase (FIGS.46A-46C).

Example 23 Wnt Treatment Prior to Transdifferentiation ImprovesTransdifferentiation Competence

Objective

The objective of this study was improve transdifferentiation competencewithin a cell population.

As described above at Example 17, active WNT signaling characterized theeGFP+ pre-disposed population. While the experiment described abovedemonstrated that induction of WNT signaling improvedtransdifferentiation efficiency when applied together with thetransdifferentiation transcription factors, it did not show whether thepre-existing WNT signaling in eGFP+ is associated with their increasedcompetence to redirect their differentiation fate.

Methods

In order to test whether WNT signaling endows the cells with competencefor transdifferentiation, eGFP+ cells were treated with 10 mM lithium(Li) for 48 hours prior to the addition of the transdifferentiationfactors. The lithium was then removed from the media when the pancreatictranscription factors were added.

Results

Upon transdifferentiation, cells that were pre-treated with Lidemonstrated an increase in insulin secretion (FIG. 48A), as well asexpression of pancreatic genes (FIG. 48B) indicating that WNT signalingis a “built-in” signal pathway enabling the cells to undergo efficienttransdifferentiation. Interestingly, endogenous PDX-1 expression levelswere not upregulated with Li pre-treatment (FIG. 48C), suggesting thatlate WNT signal is necessary for stable pancreatic repertoire.

While certain features disclosed here have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritdisclosed here.

What is claimed is:
 1. A method of manufacturing a population of humaninsulin producing cells, the method comprising the steps of: (a)obtaining adult human liver tissue; (b) processing said liver tissue torecover primary adult human liver cells; (c) propagating and expandingsaid primary adult human liver cells to a predetermined number of cells;(d) transdifferentiating said expanded cells culture; and (e) harvestingsaid transdifferentiated expanded cells; thereby manufacturing saidpopulation of human insulin producing cells having an increased insulincontent, or increased glucose regulated insulin secretion, or anycombination thereof, compared with control non-transdifferentiated livercells.
 2. The method of claim 2, wherein said glucose regulated insulinsecretion comprises at least 0.01 pg insulin/10⁶ cells/hour in responseto high glucose concentrations.
 3. The method of claim 1, whereingreater than 70% of said population of human insulin producing cellsexpress endogenous PDX-1.
 4. The method of claim 3, wherein said cellsexpressing PDX-1 also express endogenous NeuroD1 or MafA, or anycombination thereof.
 5. The method of claim 3, wherein less than 5% ofsaid population expressing PDX-1 expresses albumin and alpha-1anti-trypsin.
 6. The method of claim 1, wherein said increased insulincontent comprises an at least 5% increase compared with said controlcells.
 7. The method of claim 1, wherein at step (a) said liver tissueis obtained from a subject suffering from pancreatic or from insulindependent diabetes.
 8. The method of claim 1, wherein at step (c) saidpropagating and expanding said liver cells comprises expansion through aseries of sub-cultivation steps up to a production bioreactor system. 9.The method of claim 8, wherein said bioreactor system comprises a singlebioreactor or multiple bioreactors.
 10. The method of claim 8, whereinsaid bioreactor comprises a single use bioreactor, a multiple usebioreactor, a closed system bioreactor, or an open system bioreactor, orany combination thereof.
 11. The method of claim 1, wherein at step (d)said transdifferentiating of said expanded cells comprisestransdifferentiation through a series of bioreactor systems.
 12. Themethod of claim 1, wherein at step (d) said transdifferentiatingcomprises: (a) infecting said expanded cells with an adenoviral vectorcomprising a nucleic acid encoding a human PDX-1 polypeptide, saidinfecting at a first time period; (b) infecting said expanded cells of(a) with an adenoviral vector comprising a nucleic acid encoding a humanNeuroD1 polypeptide or Pax4 polypeptide, said infecting at a second timeperiod; and (c) infecting said expanded cells of (b) with an adenoviralvector comprising a nucleic acid encoding a human MafA polypeptide, saidinfecting at a third time period.
 13. The method of claim 1, wherein atstep (d) said transdifferentiating comprises: (a) infecting saidexpanded cells with an adenoviral vector comprising a nucleic acidencoding a human PDX-1 polypeptide and encoding a second pancreatictranscription factor polypeptide, said infecting at a first time period;and (b) infecting said expanded cells of (a) with an adenoviral vectorcomprising a nucleic acid encoding a human MafA polypeptide, saidinfecting at a second time period.
 14. The method of claim 13, whereinsaid second pancreatic transcription factor is selected from NeuroD1 andPax4.
 15. The method of claim 13, wherein said first time period andsaid second time period are concurrent.
 16. The method of claim 1,further comprising a step enriching said primary adult human liver cellsfor cells predisposed to transdifferentiation.
 17. The method of claim16, wherein said predisposed cells comprise pericentral liver cells. 18.The method of claim 16, said method further comprising incubating saidprimary adult human liver cells with lithium.
 19. The method of claim18, wherein said incubating is prior to transdifferentiation.
 20. Themethod of claim 16, wherein said predisposed cells comprise cellscomprising: (a) an active Wnt-signaling pathway; (b) a capability ofactivating the glutamine synthetase response element (GSRE); (c)increased expression of HOMER1, LAMP3, BMPR2, ITGA6, DCBLD2, THBS1, orVAMP4, or any combination thereof; (d) decreased expression of ABCB1,ITGA4, ABCB4, or PRNP, or any combination thereof; or any combinationthereof.
 21. The method of claim 1, wherein said pre-determined numberof cells comprises at least 1 billion cells.
 22. A population of humaninsulin producing cells manufactured by a method comprising the stepsof: (a) obtaining adult human liver tissue; (b) processing said livertissue to recover primary adult human primary liver cells; (c)propagating and expanding said primary adult human liver cells to apredetermined number of cells; (d) transdifferentiating said expandedcells; and (e) harvesting said transdifferentiated expanded culture;wherein said population of human insulin producing cells have anincreased insulin content or increased glucose stimulated insulinsecretion, or any combination thereof, compared with controlnon-transdifferentiated liver cells.
 23. The population of human insulinproducing cells of claim 22, wherein said glucose regulated insulinsecretion comprises at least 0.01 pg insulin/10⁶ cells/hour in responseto high glucose concentrations.
 24. The population of human insulinproducing cells of claim 22, wherein greater than 70% of said populationof human insulin producing cells express endogenous PDX-1.
 25. Thepopulation of human insulin producing cells of claim 24, wherein saidcells expressing PDX-1 also express endogenous NeuroD1 or MafA, or anycombination thereof.
 26. The population of human insulin producing cellsof claim 24, wherein less than 5% of said population expressing PDX-1also expresses albumin and alpha-1 anti-trypsin.
 27. The population ofhuman insulin producing cells of claim 22, wherein said increasedinsulin content comprises an at least 5% increase compared with saidcontrol cells.
 28. The population of human insulin producing cells ofclaim 22, for use in a cell-based therapy for a patient suffering frompancreatitis or from insulin dependent diabetes.
 29. The population ofhuman insulin producing cells of claim 28, wherein said cells areautologous or allogeneic with said patient.
 30. A composition comprisingthe population of human insulin producing cells of claim 22, and apharmaceutically acceptable carrier.